Methods of treating skin cancer by administering a pd-1 inhibitor

ABSTRACT

The present invention provides methods for treating, reducing the severity, or inhibiting the growth of cancer (e.g., skin cancer). The methods of the present invention comprise administering to a subject in need thereof a therapeutically effective amount of a programmed death 1 (PD-1) antagonist (e.g., an anti-PD-1 antibody). In certain embodiments, the skin cancer is cutaneous squamous cell carcinoma or basal cell carcinoma.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.16/559,159 filed Sep. 3, 2019, which is a continuation of U.S. patentapplication Ser. No. 15/593,915 filed May 12, 2017 (now U.S. Pat. No.10,457,725), which claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application Nos. 62/335,743, filed on May 13, 2016;62/340,142, filed on May 23, 2016; 62/348,546, filed on Jun. 10, 2016;62/350,305, filed on Jun. 15, 2016; 62/364,920, filed on Jul. 21, 2016;62/374,020, filed on Aug. 12, 2016; and 62/451,274, filed on Jan. 27,2017, the disclosures of all of which are hereby incorporated byreference in their entireties.

FIELD OF THE INVENTION

The present invention relates to methods for treating skin cancercomprising administering to a subject in need thereof a therapeuticallyeffective amount of an antibody that specifically binds to programmeddeath 1 (PD-1) receptor.

BACKGROUND OF THE INVENTION

Skin cancer is the most common cancer in the United States (Guy et al2015, Am. J. Prey. Med. 48:183-7). An estimated 5.4 million cases ofnon-melanoma skin cancer, including basal cell carcinoma and squamouscell carcinoma, were diagnosed in the United States in 2012 (Rogers etal 2015, JAMA Dermatol., Published online Apr. 30, 2015). Cutaneoussquamous cell carcinoma (CSCC) is the second-most common malignancy inthe US, after basal cell carcinoma (BCC) (Karia et al 2013, J. Am. Acad.Dermatol. 68:957-966). Risk factors for CSCC include UV exposure,advanced age, and immunosuppression (Alam et al 2001, New Engl. J. Med.344 (975-983); Madan 2010, Lancet 375: 673-685). Although the vastmajority of individuals with diagnosis of CSCC or BCC have a veryfavorable prognosis, CSCC has a greater propensity for aggressiverecurrences than BCC. Individuals diagnosed with CSCC, unlike thosediagnosed with BCC, have an increased mortality compared withage-matched controls (Rees et al 2015, Int. J. Cancer 137: 878-84).

Surgical resection is the centerpiece of clinical management of CSCC.The primary goal is complete resection of cancer, and acceptablecosmetic outcome is a secondary goal. Factors associated with poorprognosis in CSCC include tumor size >2 cm, tumor depth >2mm, perineuralinvasion, host immunosuppression, and recurrent lesions. For the smallpercentage of patients who develop unresectable locally recurrent ormetastatic disease, treatment options are limited. Patients may beadministered post-operative radiation therapy. Chemotherapy is not anattractive option for many patients due to safety and tolerabilityconcerns.

The most common clinical subtype is nodular BCC. Less common clinicalsubtypes are superficial, morphoeic (fibrosing), and fibroepithelial.Most patients are cured by surgery, but a small percentage of patientsdevelop unresectable locally advanced or metastatic disease. Virtuallyall BCCs are characterized by aberrant signaling of the hedgehogsignaling pathway, most commonly due to sporadic loss-of-functionmutation in the gene encoding protein patched homologue (PTCH), a tumorsuppressor. A PTCH mutation results in loss of patched-mediatedinhibition of the G-protein coupled receptor Smoothened (SMO), therebyenhancing downstream signaling that results in uncontrolled cellularproliferation (Sekulic et al 2016, Cell 164:831). Recognition of theoncogenic role of SMO in BCC led to the development of vismodegib andsonidegib, orally available inhibitors of SMO, generally referred to asHedgehog Inhibitors (HHIs). In addition to adverse side-effects of theHHIs, it was found that for patients that progress on one HHI(vismodegib), subsequent treatment with another HHI (sonedegib) did notresult in tumor inhibition (Danial et al 2016, Clin. Cancer Res. 22:1325-29). There is no approved agent for BCC in patients who experiencedprogression of disease on HHI therapy, or who are intolerant of priorHHI therapy.

Therefore, there is a need for safe and effective systemic therapies forskin cancer, including CSCC and BCC.

BRIEF SUMMARY OF THE INVENTION

According to certain embodiments, the present invention provides methodsfor treating or ameliorating at least one symptom or indication, orinhibiting the growth of cancer in a subject. The methods according tothis aspect of the invention comprise administering to a subject in needthereof a therapeutically effective amount of an antibody orantigen-binding fragment thereof that specifically binds to programmeddeath 1 (PD-1), optionally, in combination with radiation therapy.

According to certain embodiments, the present invention includes methodsto treat cancer including a solid tumor, the methods comprisingselecting a subject with a cancer and administering one or more doses ofan anti-PD-1 antibody in combination with one or more doses of radiationtherapy. In certain embodiments, administration of the combinationresults in enhanced therapeutic efficacy or anti-tumor efficacy ascompared to administration of either the antibody or radiation alone.

In certain embodiments of the present invention, methods are providedfor treating or ameliorating at least one symptom or indication, orinhibiting the growth of cancer in a subject. In certain embodiments ofthe present invention, methods are provided for delaying the growth of atumor or preventing tumor recurrence. In certain embodiments of thepresent invention, methods are provided for increasing the overall orprogression-free survival of a patient with cancer. The methods,according to this aspect of the invention, comprise sequentiallyadministering one or more doses of a therapeutically effective amount ofan antibody or antigen-binding fragment thereof that specifically bindsto PD-1. In one embodiment, the anti-PD-1 antibody is administered incombination with radiation therapy.

In certain embodiments, the cancer or tumor is a solid tumor ormalignancy. In certain embodiments, the solid tumor is selected from thegroup consisting of colorectal cancer, ovarian cancer, prostate cancer,breast cancer, brain cancer, cervical cancer, bladder cancer, analcancer, uterine cancer, colon cancer, liver cancer, pancreatic cancer,lung cancer, endometrial cancer, bone cancer, testicular cancer, skincancer, kidney cancer, stomach cancer, esophageal cancer, head and neckcancer, salivary gland cancer, and myeloma.

In certain embodiments, the anti-PD-1 antibody is administered as a‘first-line’ treatment to a patient with cancer, wherein the patient hasnot received prior systemic treatment for the cancer. In certainembodiments, the anti-PD-1 antibody is administered as ‘second-line’treatment to a patient with cancer (e.g., metastatic cancer), whereinthe patient has been previously treated with ‘standard-of-care’ therapyincluding, but not limited to chemotherapy, surgery and radiation.

One embodiment of the invention pertains to an anti-PD-1 antibody foruse in the treatment of skin cancer. In certain embodiments, the skincancer is a non-melanoma skin cancer including, but not limited to,cutaneous squamous cell carcinoma and basal cell carcinoma. Theanti-PD-1 antibody may be administered, as described herein, to apatient with metastatic or locally advanced cutaneous squamous cellcarcinoma. In certain embodiments, the anti-PD-1 antibody isadministered, as described herein, to a patient with advanced basal cellcarcinoma, wherein the patient is intolerant to a Hedgehog pathwayinhibitor (e.g., vismodegib, sonedegib) or has been treated with aHedgehog pathway inhibitor and shows progressive disease.

In certain embodiments, each dose of anti-PD-1 antibody comprises 0.1-20mg/kg of the subject's body weight. In certain embodiments, each dose ofanti-PD-1 antibody comprises 0.3, 1, 3, 5, or 10 mg/kg of the subject'sbody weight. In certain embodiments, each dose of the anti-PD-1 antibodycomprises 20-600 mg. In one embodiment, each dose of the anti-PD-1antibody comprises about 200 mg. In one embodiment, each dose of theanti-PD-1 antibody comprises about 250 mg. In one embodiment, each doseof the anti-PD-1 antibody comprises about 350 mg.

In certain embodiments, the radiation therapy is administered in one ormore doses. In certain embodiments, each dose of radiation therapycomprises 2-100 Gray (Gy). In certain embodiments, the radiation therapyis hypofractionated radiation therapy. In certain embodiments, theradiation therapy comprises 2-12 fractions.

In certain embodiments, the methods of the present invention compriseadministering a therapeutically effective amount of an anti-PD-1antibody prior to, concurrent with, or subsequent to radiation therapy.In one embodiment, the methods of the present invention compriseadministering an anti-PD-1 antibody prior to a dose of radiationtherapy.

In certain embodiments, the methods of the present invention compriseadministering 0-50 therapeutic doses each of an anti-PD-1 antibody,wherein each dose is administered 0.5-12 weeks after the immediatelypreceding dose. In one embodiment, each dose is administered 1 weekafter the immediately preceding dose. In one embodiment, each dose isadministered 2 weeks after the immediately preceding dose. In oneembodiment, each dose is administered 3 weeks after the immediatelypreceding dose.

In certain embodiments, the one or more doses of anti-PD-1 antibody andoptionally radiation therapy are comprised in a treatment cycle. Themethods, according to this aspect of the invention, compriseadministering to a subject in need thereof at least one treatment cyclewherein the at least one treatment cycle comprises one or more doses ofan anti-PD-1 antibody. In certain embodiments, up to 12 treatment cyclesare administered to a subject in need thereof. In certain embodiments,at least one treatment cycle further comprises one or more doses ofradiation therapy. In certain embodiments, radiation therapy isadministered in only one treatment cycle. In certain embodiments, theradiation therapy is hypofractionated radiation therapy. In certainembodiments, the anti-PD-1 antibody is administered before radiationtherapy.

In certain embodiments, the anti-PD-1 antibody and the radiation therapyare administered in combination with an additional therapeutic agent ortherapy (e.g., cyclophosphamide, or any agent or therapy disclosedherein).

In certain embodiments, the treatment produces one or more therapeuticeffects selected from the group consisting of tumor regression, abscopaleffect inhibition of tumor metastasis, reduction in metastatic lesionsover time, reduced use of chemotherapeutic or cytotoxic agents,reduction in tumor burden, increase in progression-free survival,increase in overall survival, complete response, partial response, andstable disease.

According to certain embodiments, the anti-PD-1 antibody orantigen-binding protein comprises the heavy chain complementaritydetermining regions (HCDRs) of a heavy chain variable region (HCVR)comprising the amino acid sequence of SEQ ID NO: 1 and the light chainCDRs of a light chain variable region (LCVR) comprising the amino acidsequence of SEQ ID NO: 2. One such type of antigen-binding protein thatcan be used in the context of the methods of the present invention is ananti-PD-1 antibody such as REGN2810.

In certain embodiments, the present invention provides use of ananti-PD-1 antibody or antigen-binding fragment thereof in themanufacture of a medicament to treat or inhibit the growth of cancer ina subject, including humans. In certain embodiments, the cancer is asolid tumor. In certain embodiments, the cancer is colorectal cancer,ovarian cancer, prostate cancer, breast cancer, brain cancer, cervicalcancer, bladder cancer, anal cancer, uterine cancer, colon cancer, livercancer, pancreatic cancer, lung cancer, endometrial cancer, bone cancer,testicular cancer, skin cancer, kidney cancer, stomach cancer,esophageal cancer, head and neck cancer, salivary gland cancer, ormyeloma.

In certain embodiments, the present invention provides use of ananti-PD-1 antibody or antigen-binding fragment thereof in themanufacture of a medicament in combination with radiation therapy totreat or inhibit the growth of cancer in a subject, including humans. Incertain embodiments, the cancer is a solid tumor. In certainembodiments, the cancer is colorectal cancer, ovarian cancer, prostatecancer, breast cancer, brain cancer, cervical cancer, bladder cancer,anal cancer, uterine cancer, colon cancer, liver cancer, pancreaticcancer, lung cancer, endometrial cancer, bone cancer, testicular cancer,skin cancer, kidney cancer, stomach cancer, esophageal cancer, head andneck cancer, salivary gland cancer, or myeloma.

In one aspect, the present invention provides a kit for treating asubject afflicted with a cancer, the kit comprising: (a) a dosage of anantibody or an antigen-binding portion thereof that specifically bindsto and inhibits PD-1; and (b) instructions for using the anti-PD-1antibody for treating the subject according to the methods disclosedherein. In certain embodiments, the cancer is selected from the groupconsisting of colorectal cancer, ovarian cancer, prostate cancer, breastcancer, brain cancer, cervical cancer, bladder cancer, anal cancer,uterine cancer, colon cancer, liver cancer, pancreatic cancer, lungcancer, endometrial cancer, bone cancer, testicular cancer, skin cancer,kidney cancer, stomach cancer, esophageal cancer, head and neck cancer,salivary gland cancer, and myeloma.

Other embodiments of the present invention will become apparent from areview of the ensuing detailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the study design including dosing of an anti-PD-1 antibodyand radiation (XRT) in mice implanted with MC38 tumors (study describedin Example 1 herein).

FIG. 2 shows the average tumor growth in mice treated with isotypecontrol antibody (●), anti-PD-1 antibody (▪), isotype control+radiation(XRT) (▴), or anti-PD-1 antibody+XRT (▾) in the study described inExample 1 herein.

FIG. 3 shows the overall survival of mice treated with isotype controlantibody (●), anti-PD-1 antibody (▪), isotype control+radiation (XRT)(▴), or anti-PD-1 antibody+XRT (▾) in the study described in Example 1herein.

FIG. 4 shows the study design including dosing of an anti-PD-1 antibodyand radiation (XRT) in mice implanted with B16F10.9 tumors (studydescribed in Example 2 herein).

FIG. 5 shows the average tumor growth in mice treated with isotypecontrol antibody (●), anti-PD-1 antibody (▪), isotype control+radiation(XRT) (♦), or anti-PD-1 antibody+XRT (◯) in the study described inExample 2 herein.

FIG. 6 shows the overall survival of mice treated with isotype controlantibody (●), anti-PD-1 antibody (▪), isotype control+radiation (XRT)(♦), or anti-PD-1 antibody+XRT (◯) in the study described in Example 2herein.

FIG. 7 shows the study design including dosing of an anti-PD-1 antibodyand radiation (XRT) in mice implanted with MC38 tumors (study describedin Example 4 herein)

FIG. 8 shows average primary tumor growth in mice treated with isotypecontrol antibody (●), anti-PD-1 antibody (▪), isotype control+radiation(XRT) (▴), or anti-PD-1 antibody+XRT (▾) in the study described inExample 4 herein.

FIG. 9 shows overall survival of mice treated with isotype controlantibody (▪), anti-PD-1 antibody (▪), isotype control+radiation (XRT)(▴), or anti-PD-1 antibody+XRT (▾) in the study described in Example 4herein.

FIG. 10 shows secondary tumor growth in mice treated with isotypecontrol antibody (●), anti-PD-1 antibody (▪), isotype control+radiation(XRT) (▴), or anti-PD-1 antibody+XRT (▾) in the study described inExample 4 herein.

FIG. 11 shows the study design including dosing of an anti-PD-1antibody, an anti-GITR antibody, and radiation (XRT) in mice implantedwith MC38 tumors (study described in Example 5 herein).

FIG. 12 shows the average tumor growth in mice treated with isotypecontrol antibody (●), anti-PD-1 antibody (▪), anti-GITR antibody (▴),combination of anti-PD-1 antibody and anti-GITR antibody (▾), isotypecontrol+radiation (XRT) (♦),anti-PD-1 antibody+XRT (0), anti-GITRantibody+XRT (□), or combination of anti-PD-1 antibody, anti-GITRantibody+XRT (Δ) in the study described in Example 5 herein.

FIG. 13 shows the overall survival of mice treated with isotype controlantibody (●), anti-PD-1 antibody (▪), anti-GITR antibody (▴),combination of anti-PD-1 antibody and anti-GITR antibody (▾), isotypecontrol+radiation (XRT) (♦), anti-PD-1 antibody+XRT (◯), anti-GITRantibody+XRT (▾), or combination of anti-PD-1 antibody, anti-GITRantibody+XRT (Δ) in the study described in Example 5 herein.

FIG. 14A shows a radiographic image of lung metastases in a basal cellcarcinoma (BCC) patient indicated by arrows at baseline, left, and atWeek 24, right.

FIG. 14B shows a radiographic image of neck mass in a cutaneous squamouscell carcinoma (CSCC) patient at baseline, left, and at Week 16, right.

DETAILED DESCRIPTION

Before the present invention is described, it is to be understood thatthis invention is not limited to particular methods and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. As used herein, the term“about,” when used in reference to a particular recited numerical value,means that the value may vary from the recited value by no more than 1%.For example, as used herein, the expression “about 100” includes 99 and101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice of the present invention,the preferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to describe intheir entirety.

Methods of Treating or Inhibiting Growth of Cancer

The present invention includes methods for treating, ameliorating orreducing the severity of at least one symptom or indication, orinhibiting the growth of a cancer in a subject. The methods according tothis aspect of the invention comprise administering to a subject in needthereof a therapeutically effective amount of an antibody orantigen-binding fragment thereof that specifically binds PD-1. Incertain embodiments, the anti-PD-1 antibody is administered incombination with an anti-tumor therapy (described elsewhere herein). Inone embodiment, the anti-tumor therapy is radiation therapy. As usedherein, the terms “treat”, “treating”, or the like, mean to alleviatesymptoms, eliminate the causation of symptoms either on a temporary orpermanent basis, to delay or inhibit tumor growth, to reduce tumor cellload or tumor burden, to promote tumor regression, to cause tumorshrinkage, necrosis and/or disappearance, to prevent tumor recurrence,to prevent or inhibit metastasis, to inhibit metastatic tumor growth,and/or to increase duration of survival of the subject.

As used herein, the expression “a subject in need thereof” means a humanor non-human mammal that exhibits one or more symptoms or indications ofcancer, and/or who has been diagnosed with cancer, including a solidtumor and who needs treatment for the same. In many embodiments, theterm “subject” may be interchangeably used with the term “patient”. Forexample, a human subject may be diagnosed with a primary or a metastatictumor and/or with one or more symptoms or indications including, but notlimited to, unexplained weight loss, general weakness, persistentfatigue, loss of appetite, fever, night sweats, bone pain, shortness ofbreath, swollen abdomen, chest pain/pressure, enlargement of spleen, andelevation in the level of a cancer-related biomarker (e.g., CA125). Theexpression includes subjects with primary or established tumors. Inspecific embodiments, the expression includes human subjects that haveand/or need treatment for a solid tumor, e.g., colon cancer, breastcancer, lung cancer, prostate cancer, skin cancer, liver cancer, bonecancer, ovarian cancer, cervical cancer, pancreatic cancer, head andneck cancer, and brain cancer. The term includes subjects with primaryor metastatic tumors (advanced malignancies). In certain embodiments,the expression “a subject in need thereof” includes patients with asolid tumor that is resistant to or refractory to or is inadequatelycontrolled by prior therapy (e.g., treatment with an anti-cancer agent).For example, the expression includes subjects who have been treated withone or more lines of prior therapy such as treatment with chemotherapy(e.g., carboplatin or docetaxel). In certain embodiments, the expression“a subject in need thereof” includes patients with a solid tumor whichhas been treated with one or more lines of prior therapy but which hassubsequently relapsed or metastasized. For example, patients with asolid tumor that may have received treatment with one or moreanti-cancer agents leading to tumor regression; however, subsequentlyhave relapsed with cancer resistant to the one or more anti-canceragents (e.g., chemotherapy-resistant cancer) are treated with themethods of the present invention. The expression also includes subjectswith a solid tumor for which conventional anti-cancer therapy isinadvisable, for example, due to toxic side effects. For example, theexpression includes patients who have received one or more cycles ofchemotherapy with toxic side effects.

In certain embodiments, the methods of the present invention may be usedto treat patients that show elevated levels of one or morecancer-associated biomarkers [e.g., programmed death ligand 1 (PD-L1),CA125, CA19-9, prostate-specific antigen (PSA), lactate dehydrogenase,KIT, carcinoembryonic antigen, epidermal growth factor receptor (EGFR),ALK gene rearrangement]. For example, the methods of the presentinvention comprise administering a therapeutically effective amount ofan anti-PD-1 antibody in combination with radiation therapy to a patientwith an elevated level of PD-L1 and/or EGFR. In a preferred embodiment,the methods of the present invention are used in patients with cancerthat are selected on the basis of PD-L1 expression in cancer tissue. Incertain embodiments, the methods of the present invention are used totreat patients with a cancer wherein the patients are selected on thebasis of at least 1%, at least 2%, at least 5%, at least 10%, at least20%, at least 30%, at least 40% or at least 50% PD-L1 expression incancer tissue and/or immune cells. Methods to determine PD-L1 expressionin cancer tissue and/or immune cells are well-known in the art. Incertain embodiments, the expression of PD-L1 in tumor tissue isdetermined by any assay known in the art, for example, by an ELISA assayor by an immunohistochemistry (IHC) assay, as described in PCTpublications WO2016124558 or WO2016191751 or US Patent ApplicationPublication US20160305947. In certain embodiments, the expression ofPD-L1 is determined by quantitating RNA expression, for example, by insitu hybridization or by RT-PCR. In certain embodiments, the expressionof PD-L1 is determined by imaging with a labeled anti-PD-L1 antibody,for example, by immuno-positron emission tomography or iPET [See, e.g.,The Oncologist, 12: 1379 (2007); Journal of Nuclear Medicine, 52(8):1171 (2011); US Provisional Patent Application No.: 62/428,672, filedDecember 1, 2016].

In certain embodiments, the methods of the present invention are used ina subject with a solid tumor. The terms “tumor”, “cancer” and“malignancy” are interchangeably used herein.

As used herein, the term “solid tumor” refers to an abnormal mass oftissue that usually does not contain cysts or liquid areas. Solid tumorsmay be benign (not cancer) or malignant (cancer). For the purposes ofthe present invention, the term “solid tumor” means malignant solidtumors. The term includes different types of solid tumors named for thecell types that form them, viz. sarcomas, carcinomas and lymphomas.However, the term does not include leukemias. In various embodiments,the term “solid tumor” includes cancers arising from connective orsupporting tissue (e.g., bone or muscle) (referred to as sarcomas),cancers arising from the body's glandular cells and epithelial cellswhich line body tissues (referred to as carcinomas), and cancers of thelymphoid organs such as lymph nodes, spleen and thymus (referred to aslymphomas). Lymphoid cells occur in almost all tissues of the body andtherefore, lymphomas may develop in a wide variety of organs. In certainembodiments, the term “solid tumor” includes cancers including, but notlimited to, colorectal cancer, ovarian cancer, prostate cancer, breastcancer, brain cancer, cervical cancer, bladder cancer, anal cancer,uterine cancer, colon cancer, liver cancer, pancreatic cancer, lungcancer, endometrial cancer, bone cancer, testicular cancer, skin cancer,kidney cancer, stomach cancer, esophageal cancer, head and neck cancer,salivary gland cancer, and myeloma. In certain embodiments, the term“solid tumor” includes cancers including, but not limited to,hepatocellular carcinoma, non-small cell lung cancer, head and necksquamous cell cancer, basal cell carcinoma, breast carcinoma, cutaneoussquamous cell carcinoma, chondrosarcoma, angiosarcoma,cholangiocarcinoma, soft tissue sarcoma, colorectal cancer, melanoma,Merkel cell carcinoma, and glioblastoma multiforme. In certainembodiments, the term “solid tumor” comprises more than one solid tumorlesions located separate from one another, e.g., 2, more than 2, morethan 5, more than 10, more than 15, more than 20, or more than 25lesions in a subject in need of treatment. In certain embodiments, themore than one lesions are located distally from one another in the sameorgan. In certain other embodiments, the tumor lesions may be located indifferent organs.

In certain embodiments, the present invention includes methods to treator inhibit growth of a cancer including, but not limited to, colorectalcancer, ovarian cancer, prostate cancer, breast cancer, brain cancer,cervical cancer, bladder cancer, anal cancer, uterine cancer, coloncancer, liver cancer, pancreatic cancer, lung cancer, endometrialcancer, bone cancer, testicular cancer, skin cancer, kidney cancer,stomach cancer, esophageal cancer, head and neck cancer, salivary glandcancer, and myeloma. In certain embodiments, the present inventionincludes methods to treat or inhibit the growth of a cancer including,but not limited to, hepatocellular carcinoma, non-small cell lungcancer, head and neck squamous cell cancer, basal cell carcinoma,cutaneous squamous cell carcinoma, chondrosarcoma, angiosarcoma,cholangiocarcinoma, soft tissue sarcoma, colorectal cancer, melanoma,Merkel cell carcinoma, and glioblastoma multiforme. In certainembodiments, the present invention includes methods to treat advancedsolid tumors including but not limited to, metastatic cutaneous squamouscell carcinoma (CSCC), unresectable locally advanced CSCC, metastaticcolorectal cancer, advanced or metastatic hepatocellular cancer,advanced non-small cell lung cancer, basal cell carcinoma, recurrentglioblastoma multiforme, castrate recurrent prostate cancer and anyadvanced solid tumor refractory to first-line therapy. The methods,according to this aspect, comprise administering a therapeuticallyeffective amount of an anti-PD-1 antibody. In certain embodiments, themethods comprise administering a therapeutically effective amount of ananti-PD-1 antibody in combination with an anti-tumor therapy. Anti-tumortherapies include, but are not limited to, conventional anti-tumortherapies such as chemotherapy, radiation, surgery. Other anti-tumortherapies are described elsewhere herein. In one embodiment, theanti-tumor therapy comprises radiation therapy. In certain embodiments,one or more doses of an anti-PD-1 antibody are administered to a subjectin need thereof, wherein each dose is administered 0.5, 1, 2, 3, 4, 5,6, 7, 8, 9 or 10 weeks after the immediately preceding dose. In certainembodiments, each dose comprises 0.1-10 mg/kg (e.g., 0.3 mg/kg, 1 mg/kg,3 mg/kg, or 10 mg/kg) of the subject's body weight. In certain otherembodiments, each dose comprises 20-600 mg of the anti-PD-1 antibody,e.g., 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 400 mg, or 500 mgof the anti-PD-1 antibody.

In certain embodiments, the present invention includes methods to treata cancer or inhibit the growth of a cancer with microsatelliteinstability (MSI). As used herein, the term “microsatelliteinstability,” also known as “MSI” refers to the changes inmicrosatellite repeats in tumor cells or genetic hypermutability causeddue to deficient DNA mismatch repair. Microsatellites, also known assimple sequence repeats, are repeated sequences of DNA comprisingrepeating units 1-6 base pairs in length. Although the length ofmicrosatellites is highly variable from person to person and contributesto the DNA fingerprint, each individual has microsatellites of a setlength. MSI results from the inability of the mismatch repair (MMR)proteins to fix a DNA replication error. MSI comprises DNApolymorphisms, wherein the replication errors vary in length instead ofsequence. MSI comprises frame-shift mutations, either through insertionsor deletions, or hypermethylation, leading to gene silencing. It isknown in the art that microsatellite instability may result in coloncancer, gastric cancer, endometrium cancer, ovarian cancer,hepatobiliary tract cancer, urinary tract cancer, brain cancer, and skincancers. The present invention includes methods to treat cancers withMSI, the methods comprising administering to a patient in need thereof atherapeutically effective amount of an anti-PD-1 antibody, optionally,in combination with radiation therapy.

One embodiment of the invention pertains to an anti-PD-1 antibody (e.g.,REGN2810) for use in the treatment of advanced solid tumors with MSIincluding, but not limited to metastatic colorectal cancer with MSI,metastatic endometrial cancer with MSI, and castrate recurrent prostatecancer with MSI. In certain embodiments, one or more doses of theanti-PD-1 antibody are administered to a subject with an advanced solidtumor with MSI, wherein each dose comprises 0.1 to 20 mg/kg of thesubject's body weight, and wherein each dose is administered 0.5 to 4weeks after the immediately preceding dose. In certain embodiments, oneor more doses of the anti-PD-1 antibody are administered to a subjectwith an advanced solid tumor with MSI, wherein each dose comprises20-600 mg of the anti-PD-1 antibody, and wherein each dose isadministered 0.5 to 4 weeks after the immediately preceding dose.

As used herein, the term “radiation therapy”, also referred to as “XRT”means using ionizing radiation to kill cancer cells, generally as partof anti-cancer therapy. X-rays, gamma rays or charged particles (e.g.,protons or electrons) are used to generate ionizing radiation. Radiationtherapy may be delivered by a machine placed outside the patient's body(external-beam radiation therapy), or by a source placed inside apatient's body (internal radiation therapy or brachytherapy), or throughsystemic radioisotopes delivered intravenously or orally (systemicradioisotope therapy). Radiation therapy may be planned and administeredin conjunction with imaging-based techniques such a computed tomography(CT), magnetic resonance imaging (MRI) to accurately determine the doseand location of radiation to be administered. In various embodiments,radiation therapy is selected from the group consisting of totalall-body radiation therapy, conventional external beam radiationtherapy, stereotactic radiosurgery, stereotactic body radiation therapy,3-D conformal radiation therapy, intensity-modulated radiation therapy,image-guided radiation therapy, tomotherapy, brachytherapy, and systemicradiation therapy. Depending upon the intent, in certain embodiments,radiation therapy is curative, adjuvinating or palliative. In specificembodiments, the term “radiation therapy” refers to hypofractionatedradiation therapy. Hypofractionated radiation therapy refers toradiation therapy in which a radiation dose is comprised in 2 or morefractions. In various embodiments, each fraction comprises 2-20 Gy. Forexample, a radiation dose of 50 Gy may be split up into 10 fractions,each comprising 5 Gy. In certain embodiments, the 2 or more fractionsare administered on consecutive or sequential days. In certain otherembodiments, the 2 or more fractions are administered once in 2 days,once in 3 days, once in 4 days, once in 5 days, once in 6 days, once in7 days, or in a combination thereof.

According to certain embodiments, the present invention includes methodsfor treating, or delaying or inhibiting the growth of a tumor. Incertain embodiments, the present invention includes methods to promotetumor regression. In certain embodiments, the present invention includesmethods to reduce tumor cell load or to reduce tumor burden. In certainembodiments, the present invention includes methods to prevent tumorrecurrence. The methods, according to this aspect of the invention,comprise sequentially administering a therapeutically effective amountof an anti-PD-1 antibody in combination with radiation therapy to asubject in need thereof, wherein the antibody is administered to thesubject in multiple doses, e.g., as part of a specific therapeuticdosing regimen. For example, the therapeutic dosing regimen may compriseadministering one or more doses of an anti-PD-1 antibody to the subjectat a frequency of about once a day, once every two days, once everythree days, once every four days, once every five days, once every sixdays, once a week, once every two weeks, once every three weeks, onceevery four weeks, once a month, once every two months, once every threemonths, once every four months, or less frequently. In certainembodiments, the one or more doses of anti-PD-1 antibody areadministered in combination with one or more doses of radiation therapy,wherein the one or more doses of radiation are administered to thesubject at a frequency of about once a day, once every two days, onceevery three days, once every four days, once every five days, once everysix days, once a week, once every two weeks, once every three weeks,once every four weeks, once a month, once every two months, once everythree months, once every four months, or less frequently.

In certain embodiments, the one or more doses are comprised in atreatment cycle. The methods, according to this aspect, compriseadministering to a subject in need thereof at least one treatment cycle,wherein the at least one treatment cycle comprises 1-10 doses of ananti-PD-1 antibody and optionally one or more doses of radiationtherapy. In certain embodiments, 2-12 treatment cycles are administeredto a subject in need thereof.

In specific embodiments, the present invention provides methods forincreased anti-tumor efficacy or increased tumor inhibition. Themethods, according to this aspect of the invention, compriseadministering to a subject with a solid tumor a therapeuticallyeffective amount of an anti-PD-1 antibody prior to administering aradiation dose, wherein the anti-PD-1 antibody may be administered about1 day, more than 1 day, more than 2 days, more than 3 days, more than 4days, more than 5 days, more than 6 days, more than 7 days, or more than8 days prior to the radiation therapy. In certain embodiments, themethods provide for increased tumor inhibition, e.g., by about 20%, morethan 20%, more than 30%, more than 40% more than 50%, more than 60%,more than 70% or more than 80% as compared to a subject administeredwith a radiation dose prior to the anti-PD-1 antibody. In certainembodiments, the radiation therapy comprises hypofractionated radiationtherapy.

In certain embodiments, the present invention provides methods fortreating cancer, the methods comprising selecting a subject with a firsttumor lesion and at least a second tumor lesion and administering one ormore doses of an anti-PD-1 antibody in combination with radiationtherapy such that both the lesions are treated. In specific embodiments,the methods comprise administering radiation therapy to the first tumorlesion but not the second tumor lesion wherein the administration leadsto tumor regression in both the tumor lesions (abscopal effect). Incertain embodiments, the methods comprising selecting a subject with afirst tumor lesion and at least a second tumor lesion and administeringone or more doses of an anti-PD-1 antibody in combination withhypofractionated radiation therapy wherein the hypofractionatedradiation therapy is administered to the first lesion but not the secondlesion and wherein both the lesions are treated upon suchadministration. In certain embodiments, the anti-PD-1 antibody isadministered before radiation therapy.

In certain embodiments, the present invention includes methods fortreating cancer, the methods comprising administering to a subject inneed thereof one or more sub-therapeutic doses of an anti-PD-1 antibodyin combination with one or more anti-tumor therapies, e.g., radiationtherapy. As defined elsewhere herein, the term “sub-therapeutic dose”refers to a dose less than a therapeutic dose and may be used to reducetoxicity of the administered therapy. In certain embodiments,administering a sub-therapeutic dose of an anti-PD-1 antibody incombination with radiation therapy results in therapeutic anti-tumorefficacy as compared to administration of the sub-therapeutic dose ofthe anti-PD-1 antibody alone. In certain other embodiments, the methodsof the present invention comprise administering a therapeuticallyeffective amount of an anti-PD-1 antibody in combination with asub-therapeutic dose of an anti-tumor therapy such as chemotherapy orradiation. For example, a therapeutically effective amount of ananti-PD-1 antibody may be administered in combination with asub-therapeutic dose of cyclophosphamide, for increased efficacy ascompared to either monotherapy.

In certain embodiments, the present invention includes methods toinhibit, retard or stop tumor metastasis or tumor infiltration intoperipheral organs. The methods, according to this aspect, compriseadministering a therapeutically effective amount of an anti-PD-1antibody to a subject in need thereof. In certain embodiments, theanti-PD-1 antibody is administered in combination with radiation. In oneembodiment, the radiation is hypofractionated radiation. In oneembodiment, the radiation is administered after administering one ormore doses of the anti-PD-1 antibody.

In certain embodiments, the methods of the present invention compriseadministering a therapeutically effective amount of anti-PD-1 antibodyto a subject with advanced solid tumors. In specific embodiments, theadvanced solid tumor is metastatic lung cancer, head and neck cancer,hepatocellular cancer, or breast cancer. In certain other embodiments,the advanced solid tumor is cutaneous squamous cell cancer. In certainembodiments, the advanced solid tumor is indolent or aggressive. Incertain embodiments, the subject is not responsive to prior therapy orhas relapsed after prior therapy (e.g., with carboplatin). In certainembodiments, the subject has an advanced solid tumor that is refractoryto first line chemotherapy. In certain further embodiments, the methodsof the present invention further comprise administering radiation and/orcyclophosphamide to a subject with an advanced solid tumor.

In certain embodiments, the present invention includes methods to treator inhibit growth of a cancer including, but not limited to, colorectalcancer, ovarian cancer, prostate cancer, breast cancer, brain cancer,cervical cancer, bladder cancer, anal cancer, uterine cancer, coloncancer, liver cancer, pancreatic cancer, lung cancer, endometrialcancer, bone cancer, testicular cancer, skin cancer, kidney cancer,stomach cancer, esophageal cancer, head and neck cancer, salivary glandcancer, and myeloma. In certain embodiments, the present inventionincludes methods to treat or inhibit the growth of a cancer including,but not limited to, hepatocellular carcinoma, non-small cell lungcancer, head and neck squamous cell cancer, basal cell carcinoma,cutaneous squamous cell carcinoma, chondrosarcoma, angiosarcoma,cholangiocarcinoma, soft tissue sarcoma, colorectal cancer, melanoma,Merkel cell carcinoma, and glioblastoma multiforme. In certainembodiments, the present invention includes methods to treat advancedsolid tumors including but not limited to, metastatic cutaneous squamouscell carcinoma (CSCC), unresectable locally advanced CSCC, metastaticcolorectal cancer, advanced or metastatic hepatocellular cancer,advanced non-small cell lung cancer, recurrent glioblastoma multiforme,newly diagnosed glioblastoma multiforme, castrate recurrent prostatecancer and any advanced solid tumor refractory to first-line therapy.

According to one aspect, the present invention includes methods to treator inhibit the growth of a tumor, the methods comprising: (a) selectinga patient with cutaneous squamous cell carcinoma (CSCC) wherein thepatient is selected based on an attribute selected from the groupconsisting of: (i) the patient has locally advanced CSCC; (ii) thepatient has metastatic CSCC; (iii) the tumor is unresectable; (iv) thepatient has been earlier treated with at least one anti-tumor therapy;(v) the patient has disease that is considered inoperable; (vi) surgeryand/or radiation is contraindicated; (vii) the patient has been earliertreated with radiation and the tumor is resistant or unresponsive toradiation; (viii) the patient has locally advanced CSCC and is notamenable to curative surgery; (ix) the tumor comprises uv-induced DNAdamage; and (x) the patient shows 1%, ≥5%, or ≥10% PD-L1 expression intumor cells; and (b) administering a therapeutically effective amount ofan anti-PD-1 antibody to the patient need thereof. In certainembodiments, one or more doses of the anti-PD-1 antibody areadministered 1-12 weeks after the immediately preceding dose, forexample, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 weeks after theimmediately preceding dose. In certain embodiments, each dose of theanti-PD-1 antibody comprises 0.1, 1, 0.3, 3, 4, 5, 6, 7, 8, 9 or 10mg/kg of the patient's body weight. In certain embodiments, each dosecomprises 50-500 mg of the anti-PD-1 antibody, for example 200 mg, 250mg or 350 mg of the anti-PD-1 antibody, wherein each dose isadministered 0.5, 1, 2, 3 or 4 weeks after the immediately precedingdose. In one embodiment, the anti-PD-1 antibody is REGN2810.

According to one aspect, the present invention includes methods to treator inhibit the growth of a tumor, the methods comprising: (a) selectinga patient with basal cell carcinoma (BCC) wherein the patient isselected based on an attribute selected from the group consisting of:(i) the patient has locally advanced BCC; (ii) the patient hasmetastatic BCC; (iii) the tumor is unresectable; (iv) the patient hasbeen earlier treated with at least one anti-tumor therapy; (v) thepatient has been treated earlier and progressed upon treatment with aHedgehog pathway inhibitor (e.g., vismodegib, sonedegib); (vi) thepatient is intolerant to a Hedgehog pathway inhibitor; (vii) the patienthas disease that is considered inoperable or is not amenable to curativesurgery; (viii) surgery and/or radiation is contraindicated; (ix) thepatient has been earlier treated with radiation and the tumor isresistant or unresponsive to radiation; (viii) the patient shows 1%, 5%,or 10% PD-L1 expression in tumor cells; and (ix) the tumor comprisesuv-induced DNA damage; and (b) administering a therapeutically effectiveamount of an anti-PD-1 antibody to the patient need thereof. In certainembodiments, one or more doses of the anti-PD-1 antibody areadministered 1 -12 weeks after the immediately preceding dose, forexample, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 weeks after theimmediately preceding dose. In certain embodiments, each dose of theanti-PD-1 antibody comprises 0.1, 1, 0.3, 3, 4, 5, 6, 7, 8, 9 or 10mg/kg of the patient's body weight. In certain embodiments, each dosecomprises 50-500 mg of the anti-PD-1 antibody, for example 200 mg, 250mg or 350 mg of the anti-PD-1 antibody, wherein each dose isadministered 0.5, 1, 2, 3 or 4 weeks after the immediately precedingdose. In one embodiment, the anti-PD-1 antibody is REGN2810.

In certain embodiments, each dose of the anti-PD-1 antibody isadministered 1 week, 2 weeks, 3 weeks, or 4 weeks after the immediatelypreceding dose, wherein each dose comprises 50-600 mg of the anti-PD-1antibody. In one embodiment, each dose comprises 200, 250, 300 or 350 mgof the anti-PD-1 antibody.

One embodiment of the invention pertains to an anti-PD-1 antibody (e.g.,REGN2810) for use in the treatment of cholangiocarcinoma. In certainembodiments, one or more doses of the anti-PD-1 antibody areadministered to a subject with cholangiocarcinoma, wherein each dosecomprises 0.1 to 20 mg/kg of the subject's body weight, and wherein eachdose is administered 0.5 to 4 weeks after the immediately precedingdose. In certain embodiments, each dose comprises 50-500 mg of theanti-PD-1 antibody, for example 200 mg, 250 mg or 350 mg of theanti-PD-1 antibody, wherein each dose is administered 0.5, 1, 2, 3 or 4weeks after the immediately preceding dose.

One embodiment of the invention pertains to an anti-PD-1 antibody (e.g.,REGN2810) for use in the treatment of advanced hepatocellular cancer(HCC). In certain embodiments, one or more doses of the anti-PD-1antibody are administered to a subject with HCC, wherein each dosecomprises 0.1 to 20 mg/kg of the subject's body weight, and wherein eachdose is administered 0.5 to 4 weeks after the immediately precedingdose. In certain embodiments, each dose comprises 50 — 500 mg of theanti-PD-1 antibody, for example 200 mg, 250 mg or 350 mg of theanti-PD-1 antibody, wherein each dose is administered 0.5, 1, 2, 3 or 4weeks after the immediately preceding dose.

One embodiment of the invention pertains to an anti-PD-1 antibody (e.g.,REGN2810) for use in the treatment of soft tissue sarcoma. In certainembodiments, one or more doses of the anti-PD-1 antibody areadministered to a subject with soft tissue sarcoma, wherein each dosecomprises 0.1 to 20 mg/kg of the subject's body weight, and wherein eachdose is administered 0.5 to 4 weeks after the immediately precedingdose. In certain embodiments, each dose comprises 50-500 mg of theanti-PD-1 antibody, for example 200 mg, 250 mg or 350 mg of theanti-PD-1 antibody, wherein each dose is administered 0.5, 1, 2, 3 or 4weeks after the immediately preceding dose.

One embodiment of the invention pertains to an anti-PD-1 antibody (e.g.,REGN2810) for use in the treatment of non-small cell lung cancer(NSCLC). In certain embodiments, one or more doses of the anti-PD-1antibody are administered to a subject with NSCLC, wherein each dosecomprises 0.1 to 20 mg/kg of the subject's body weight, and wherein eachdose is administered 0.5 to 4 weeks after the immediately precedingdose. In certain embodiments, one or more doses of the anti-PD-1antibody are administered to a subject with NSCLC, wherein each dosecomprises 50-600 mg of the anti-PD-1 antibody, and wherein each dose isadministered 0.5 to 4 weeks after the immediately preceding dose.

According to one aspect, the present invention includes methods to treator inhibit the growth of a tumor, the methods comprising selecting asubject with a brain cancer and administering a therapeuticallyeffective amount of an anti-PD-1 antibody or antigen-binding fragmentthereof to the subject in need thereof. In certain embodiments, thebrain cancer is glioblastoma multiforme. In one embodiment, the subjecthas newly diagnosed glioblastoma multiforme. In one embodiment, thesubject is 65 years of age. In one embodiment, the anti-PD-1 antibody isadministered as one or more doses, wherein each dose is administered 0.5to 4 weeks after the immediately preceding dose. In one embodiment, eachdose of the anti-PD-1 antibody comprises 1, 3 or 10 mg/kg of thesubject's body weight. In certain embodiments, the anti-PD-1 antibody isadministered in combination with radiation therapy. In one embodiment,the radiation therapy is hypofractionated radiation therapy. In oneembodiment, the subject is administered 20-60 Gy in 2-20 fractions. Incertain embodiments, the one or more doses of anti-PD-1 antibody arecomprised in one or more cycles of treatment, wherein each cycle oftreatment comprises 1-6 doses of the anti-PD-1 antibody. In oneembodiment, at least one cycle of treatment further comprises radiationtherapy. In a further embodiment, the radiation therapy ishypofractionated radiation therapy. In certain embodiments, the subjectis administered hypofractionated radiation therapy in the first cycle oftreatment, wherein the hypofractionated radiation therapy comprises20-60 Gy in 2-20 fractions. In one embodiment, the subject isadministered hypofractionated radiation therapy one week after theadministration of the anti-PD-1 antibody in the first cycle oftreatment. In certain embodiments, the methods of the present inventionfurther comprise administering an anti-angiogenic agent to the subjectif the subject develops intracranial edema following administration ofthe anti-PD-1 antibody. In one embodiment, the anti-angiogenic agent isa vascular endothelial growth factor (VEGF) inhibitor. In oneembodiment, the anti-angiogenic agent is an angiopoietin-2 (Ang-2)inhibitor (e.g., an anti-Ang-2 antibody such as nesvacumab). In certainembodiments, the VEGF inhibitor is selected from the group consisting ofa VEGF-inhibiting fusion protein (e.g., a “VEGF-Trap” such asaflibercept or other VEGF-inhibiting fusion protein as set forth in US7,087,411), an anti-VEGF antibody (e.g., bevacizumab), and a smallmolecule kinase inhibitor of VEGF receptor (e.g., sunitinib, sorafenib,or pazopanib).

In certain embodiments, the methods of the present invention compriseadministering an anti-PD-1 antibody in combination with radiationtherapy to a subject in need thereof as a “first line” treatment (e.g.,initial treatment). In other embodiments, an anti-PD-1 antibody incombination with radiation therapy is administered as a “second line”treatment (e.g., after prior therapy). For example, an anti-PD-1antibody in combination with radiation therapy is administered as a“second line” treatment to a subject that has relapsed after priortherapy with, e.g., chemotherapy.

The methods of the present invention, according to certain embodiments,comprise administering to a subject a therapeutically effective amountof an anti-PD-1 antibody and radiation in combination with an additionaltherapeutic agent or therapeutic regimen or procedure. The additionaltherapeutic agent or therapeutic regimen or procedure may beadministered for increasing anti-tumor efficacy, for reducing toxiceffects of one or more therapies and/or reducing the dosage of one ormore therapies. In various embodiments, the additional therapeutic agentor therapeutic regimen or procedure is selected from the groupconsisting of, e.g., chemotherapy, cyclophosphamide, surgery, a cancervaccine, a programmed death ligand 1 (PD-L1) inhibitor (e.g., ananti-PD-L1 antibody), a lymphocyte activation gene 3 (LAG3) inhibitor(e.g., an anti-LAG3 antibody), a cytotoxic T-lymphocyte-associatedprotein 4 (CTLA-4) inhibitor (e.g., ipilimumab), aglucocorticoid-induced tumor necrosis factor receptor (GITR) inhibitor(e.g., an anti-GITR antibody), a T-cell immunoglobulin and mucincontaining -3 (TIM3) inhibitor, a B- and T-lymphocyte attenuator (BTLA)inhibitor, a T cell immunoreceptor with Ig and ITIM domains (TIGIT)inhibitor, a CD47 inhibitor, an indoleamine-2,3-dioxygenase (IDO)inhibitor, a vascular endothelial growth factor (VEGF) antagonist, anangiopoietin-2 (Ang2) inhibitor, a transforming growth factor beta(TGFβ) inhibitor, an epidermal growth factor receptor (EGFR) inhibitor,an antibody to a tumor-specific antigen [e.g., CA9, CA125,melanoma-associated antigen 3 (MAGE3), carcinoembryonic antigen (CEA),vimentin, tumor-M2-PK, prostate-specific antigen (PSA), mucin-1, MART-1,and CA19-9], an anti-CD3/anti-CD20 bispecific antibody, a vaccine (e.g.,Bacillus Calmette-Guerin), granulocyte-macrophage colony-stimulatingfactor, a cytotoxin, a chemotherapeutic agent, an IL-6R inhibitor, anIL-4R inhibitor, an IL-10 inhibitor, a cytokine such as IL-2, IL-7,IL-21, and IL-15, an anti-inflammatory drug such as corticosteroids, andnon-steroidal anti-inflammatory drugs, and a dietary supplement such asanti-oxidants. In certain embodiments, the anti-PD-1 antibody may beadministered in combination with therapy including a chemotherapeuticagent, and surgery. As used herein, the phrase “in combination with”means that the anti-PD-1 antibody is administered to the subject at thesame time as, just before, or just after administration of radiationtherapy and the additional therapeutic agent. In certain embodiments,the additional therapeutic agent is administered as a co-formulationwith the anti-PD-1 antibody.

One embodiment of the invention pertains to a combination of ananti-PD-1 antibody (e.g., REGN2810), radiation therapy, cyclophosphamideand GM-CSF for use in the treatment of head and neck squamous cellcarcinoma (HNSCC). In certain embodiments, one or more doses of theanti-PD-1 antibody are administered to a subject with HNSCC, whereineach dose comprises 0.1 to 20 mg/kg of the subject's body weight, andwherein each dose is administered 0.5 to 4 weeks after the immediatelypreceding dose. In certain embodiments, each dose comprises 50-500 mg ofthe anti-PD-1 antibody, for example 200 mg, 250 mg or 350 mg of theanti-PD-1 antibody, wherein each dose is administered 0.5, 1, 2, 3 or 4weeks after the immediately preceding dose.

One embodiment of the invention pertains to a combination of ananti-PD-1 antibody (e.g., REGN2810), radiation therapy, andcyclophosphamide for use in the treatment of breast cancer. In certainembodiments, one or more doses of the anti-PD-1 antibody areadministered to a subject with breast cancer, wherein each dosecomprises 0.1 to 20 mg/kg of the subject's body weight, and wherein eachdose is administered 0.5 to 4 weeks after the immediately precedingdose.

One embodiment of the invention pertains to a combination of ananti-PD-1 antibody (e.g., REGN2810), radiation therapy, cyclophosphamideand GM-CSF for use in the treatment of advanced solid tumors in patientsthat have been previously treated with an anti-PD-1 antibody or ananti-PD-L1 antibody. In certain embodiments, one or more doses of theanti-PD-1 antibody are administered to a patient in need thereof,wherein each dose comprises 0.1 to 20 mg/kg of the subject's bodyweight, and wherein each dose is administered 0.5 to 4 weeks after theimmediately preceding dose.

One embodiment of the invention pertains to a combination of ananti-PD-1 antibody (e.g., REGN2810), docetaxel, and optionally,carboplatin for use in the treatment of advanced solid tumors that arerefractory to first-line chemotherapy. In certain embodiments, thedocetaxel is administered at a low dose. In certain embodiments, one ormore doses of the anti-PD-1 antibody are administered to a subject inneed thereof, wherein each dose comprises 0.1 to 20 mg/kg of thesubject's body weight, and wherein each dose is administered 0.5 to 4weeks after the immediately preceding dose.

One embodiment of the invention pertains to a combination of ananti-PD-1 antibody (e.g., REGN2810), and radiation therapy for use inthe treatment of newly diagnosed, or recurrent glioblastoma multiforme(GBM). In certain embodiments, one or more doses of the anti-PD-1antibody are administered to a subject in need thereof, wherein eachdose comprises 0.1 to 20 mg/kg of the subject's body weight, and whereineach dose is administered 0.5 to 4 weeks after the immediately precedingdose. In certain embodiments, the radiation is hypofractionatedradiation therapy as described herein.

Certain embodiments of the invention pertain to a combination of ananti-PD-1 antibody (e.g., REGN2810), and radiation therapy for use inthe treatment of cervix squamous cell carcinoma, anal squamous cellcarcinoma, Merkel cell carcinoma, small intestine adenocarcinoma orovarian serous carcinoma. In certain embodiments, one or more doses ofthe anti-PD-1 antibody are administered to a subject in need thereof,wherein each dose comprises 0.1 to 20 mg/kg of the subject's bodyweight, and wherein each dose is administered 0.5 to 4 weeks after theimmediately preceding dose. In certain embodiments, the radiation ishypofractionated radiation therapy as described herein.

In certain embodiments, the present invention includes methods fortreating large tumors or advanced malignancies, the methods comprisingadministering to a subject in need thereof an anti-PD-1 antibody incombination with radiation therapy and an additional therapeutic agent,wherein the additional therapeutic agent is administered to overcomeregulatory T cell (Treg)-mediated immunosuppression. In certainembodiments, the additional therapeutic agent is selected from the groupconsisting of an anti-GITR antibody, an anti-LAG3 antibody,cyclophosphamide, and GM-CSF.

As used herein, the term “large tumor” refers to the size of the tumor.It typically correlates with higher tumor burden or tumor load. Incertain embodiments, it correlates with stage of the disease, e.g.,advanced malignancy. In certain embodiments, it correlates withincreased probability of metastasis.

In certain embodiments, the present invention includes methodscomprising administering one or more doses of an anti-PD-1 antibody incombination with radiation therapy and a sub-therapeutic dose ofcyclophosphamide. As used herein, a sub-therapeutic dose ofcyclophosphamide (also referred to herein as “low-dosecyclophosphamide”) means an amount of cyclophosphamide that by itselfdoes not impart a therapeutic effect and preferably does not causetoxicity. Exemplary doses of cyclophosphamide that are considered“sub-therapeutic” in the context of the present invention include 100mg/m2, 90 mg/m2, 80 mg/m2, or less.

In one aspect, the present invention includes methods comprisingadministering a therapeutically effective amount of an anti-PD-1antibody in combination with radiation to a subject who is on abackground anti-cancer therapeutic regimen. The background anti-cancertherapeutic regimen may comprise a course of administration of, e.g., achemotherapeutic agent. The anti-PD-1 antibody in combination withradiation therapy may be added on top of the background anti-cancertherapeutic regimen. In some embodiments, the anti-PD-1 antibody isadded as part of a “background step-down” scheme, wherein the backgroundanti-cancer therapy is gradually withdrawn from the subject over time(e.g., in a stepwise fashion) while the anti-PD-1 antibody isadministered to the subject at a constant dose, or at an increasingdose, or at a decreasing dose, over time. For example, the backgroundanti-cancer therapy may comprise a chemotherapeutic agent which may beadministered at a low dose or at a subtherapeutic dose. In certainembodiments, the present invention includes methods for treating cancer,the methods comprising administering one or more doses of an anti-PD-1antibody in combination with radiation therapy and one or more doses ofa chemotherapeutic agent, wherein the chemotherapeutic agent isadministered at a subtherapeutic dose.

In certain embodiments, the radiation therapy is administered to a firsttumor lesion, but not to a second tumor lesion, wherein theadministration in combination with the anti-PD-1 antibody leads to tumorregression in both the first and second tumor lesions (abscopal effect).In certain embodiments, the methods of the present invention compriseadministering an anti-PD-1 antibody in combination with radiationtherapy to generate prolonged abscopal effect.

In certain embodiments, the methods of the present invention compriseadministering to a subject in need thereof a therapeutically effectiveamount of an anti-PD-1 antibody, optionally, in combination withradiation therapy, wherein administration of the combination leads toincreased inhibition of tumor growth. In certain embodiments, tumorgrowth is inhibited by at least about 10%, about 20%, about 30%, about40%, about 50%, about 60%, about 70% or about 80% as compared to anuntreated subject or a subject administered with either antibody orradiation as monotherapy. In certain embodiments, the administration ofan anti-PD-1 antibody and/or radiation therapy leads to increased tumorregression, tumor shrinkage and/or disappearance. In certainembodiments, the administration of an anti-PD-1 antibody and/orradiation therapy leads to delay in tumor growth and development, e.g.,tumor growth may be delayed by about 3 days, more than 3 days, about 7days, more than 7 days, more than 15 days, more than 1 month, more than3 months, more than 6 months, more than 1 year, more than 2 years, ormore than 3 years as compared to an untreated subject or a subjecttreated with either antibody or radiation as monotherapy. In certainembodiments, administration of an anti-PD-1 antibody in combination withradiation therapy prevents tumor recurrence and/or increases duration ofsurvival of the subject, e.g., increases duration of survival by morethan 15 days, more than 1 month, more than 3 months, more than 6 months,more than 12 months, more than 18 months, more than 24 months, more than36 months, or more than 48 months than an untreated subject or a subjectwhich is administered either antibody or radiation as monotherapy. Incertain embodiments, administration of the anti-PD-1 antibody incombination with radiation therapy increases progression-free survivalor overall survival. In certain embodiments, administration of ananti-PD-1 antibody in combination with radiation therapy increasesresponse and duration of response in a subject, e.g., by more than 2%,more than 3%, more than 4%, more than 5%, more than 6%, more than 7%,more than 8%, more than 9%, more than 10%, more than 20%, more than 30%,more than 40% or more than 50% over an untreated subject or a subjectwhich has received either antibody or radiation as monotherapy. Incertain embodiments, administration of an anti-PD-1 antibody and/orradiation therapy to a subject with a cancer leads to completedisappearance of all evidence of tumor cells (“complete response”). Incertain embodiments, administration of an anti-PD-1 antibody and/orradiation therapy to a subject with a cancer leads to at least 30% ormore decrease in tumor cells or tumor size (“partial response”). Incertain embodiments, administration of an anti-PD-1 antibody and/orradiation therapy to a subject with a cancer leads to complete orpartial disappearance of tumor cells/lesions including new measurablelesions. Tumor reduction can be measured by any of the methods known inthe art, e.g., X-rays, positron emission tomography (PET), computedtomography (CT), magnetic resonance imaging (MRI), cytology, histology,or molecular genetic analyses.

In certain embodiments, the methods of the present invention compriseadministering to a subject in need thereof a therapeutically effectiveamount of an anti-PD-1 antibody, wherein administration of the anti-PD-1antibody leads to increased overall survival (OS) or progression-freesurvival (PFS) of the patient as compared to a patient administered witha ‘standard-of-care’ (SOC) therapy (e.g., chemotherapy, surgery orradiation). In certain embodiments, the PFS is increased by at least onemonth, at least 2 months, at least 3 months, at least 4 months, at least5 months, at least 6 months, at least 7 months, at least 8 months, atleast 9 months, at least 10 months, at least 11 months, at least 1 year,at least 2 years, or at least 3 years as compared to a patientadministered with any one or more SOC therapies. In certain embodiments,the OS is increased by at least one month, at least 2 months, at least 3months, at least 4 months, at least 5 months, at least 6 months, atleast 7 months, at least 8 months, at least 9 months, at least 10months, at least 11 months, at least 1 year, at least 2 years, or atleast 3 years as compared to a patient administered with any one or moreSOC therapies.

The present invention also provides kits comprising an anti-PD-1antibody for therapeutic uses. Kits typically include a label indicatingthe intended use of the contents of the kit and instructions for use.The term label includes any writing, or recorded material supplied on orwith the kit, or which otherwise accompanies the kit. Accordingly, thisdisclosure provides a kit for treating a subject afflicted with acancer, the kit comprising: (a) a dosage of an antibody or anantigen-binding portion thereof that specifically binds to PD-1 andinhibits PD-1 activity; and (b) instructions for using the anti-PD-1antibody in any of the therapy methods disclosed herein. In certainembodiments for treating human patients, the kit comprises an anti-humanPD-1 antibody disclosed herein, e.g., REGN2810. In other embodiments,the anti-PD-1 antibody may be any one of nivolumab, pembrolizumab, orany of the anti-PD-1 antibodies disclosed herein. In certainembodiments, the dosage of the anti-PD-1 antibody ranges from 0.1 to 10mg/kg body weight. In certain embodiments, the dosage of the anti-PD-1antibody comprises from 50 to 600 mg.

Methods for Suppressing T regulatory Cells

According to certain aspects, the present invention provides methods forsuppressing or inhibiting the activation and/or proliferation of Tregulatory (Treg) cells. In certain embodiments, the present inventionprovides methods for suppressing the activity of Treg cells. Themethods, according to these aspects, comprise selecting a subject with asolid tumor and administering to the subject an anti-PD-1 antibody orantigen-binding fragment thereof in combination with at least one of (i)radiation therapy, and (ii) a glucocorticoid-induced tumor necrosisfactor receptor (GITR) antagonist. In certain embodiments, the methodscomprise administering to a subject in need thereof an anti-PD-1antibody or antigen-binding fragment thereof in combination withradiation therapy and a GITR antagonist.

In certain embodiments, the GITR antagonist is an anti-GITR antibody orantigen-binding fragment thereof. According to certain exemplaryembodiments of the present invention, the anti-GITR antibody, orantigen-binding fragment thereof comprises a heavy chain variable region(HCVR), light chain variable region (LCVR), and/or complementaritydetermining regions (CDRs) comprising the amino acid sequences of any ofthe anti-GITR antibodies as set forth in U.S. Ser. No. 62/256,922 (filedNov. 18, 2015), the contents of which are incorporated herein in theirentirety. Other anti-GITR antibodies that can be used in the context ofthe methods of the present invention include any of the anti-GITRantibodies as set forth in e.g., U.S. Pat. Nos. 9,228,016, 8,709,424,8,591,886, 7,812,135, or US Patent Publication No. 20150368349.

In certain embodiments, the present invention provides methods forsuppressing or eliminating Treg activity, the methods comprisingadministering to a subject in need thereof an anti-PD-1 antibody orantigen-binding fragment thereof in combination with one or more dosesof radiation and a cytotoxic T-lymphocyte antigen-4 (CTLA) antagonist.In certain embodiments, the CTLA antagonist is an anti-CTLA antibody(e.g., ipilimumab).

In certain embodiments, the present invention provides methods forsuppressing or eliminating Treg activity, the methods comprisingadministering to a subject in need thereof an anti-PD-1 antibody orantigen-binding fragment thereof in combination with one or more dosesof radiation and a lymphocyte activation gene 3 (LAG-3) antagonist. Incertain embodiments, the LAG-3 antagonist is an anti-LAG-3 antibody.Anti-LAG-3 antibodies that can be used in the context of the methods ofthe present invention are disclosed in U.S. Ser. No. 15/289,032 (filedOct. 7, 2016), the contents of which are incorporated herein in theirentirety

In certain embodiments, the present invention provides methods forsuppressing or eliminating Treg activity, the methods comprisingadministering to a subject in need thereof an anti-PD-1 antibody orantigen-binding fragment thereof in combination with one or more dosesof radiation and cyclophosphamide.

In one aspect, the methods of the present invention compriseadministration of an anti-PD-1 antibody in combination with radiationtherapy and an additional therapeutic agent selected from the groupconsisting of a GITR antagonist, an anti-LAG-3 antibody, andcyclophosphamide to a subject with a solid tumor, wherein theadministration results in an effect selected from the group consistingof inhibition of tumor growth, reduction in the size of a tumor, delayin tumor growth, inhibition of tumor metastasis, reduction in metastaticlesions over time, reduced use of chemotherapeutic or cytotoxic agents,increased survival, complete response, partial response, and stabledisease. In certain embodiments, the administration results in reductionof tumor burden in the subject. In certain embodiments, the subject hasa large tumor. As defined elsewhere herein, the term “large tumor”refers to the size of the tumor and is correlated with increased tumorburden and increased probability of occurrence of metastasis. In certainembodiments, the term refers to an advanced malignancy.

Anti-PD-1 Antibodies and Antigen-Binding Fragments Thereof

According to certain exemplary embodiments of the present invention, themethods comprise administering a therapeutically effective amount of ananti-PD-1 antibody or antigen-binding fragment thereof. The term“antibody,” as used herein, includes immunoglobulin molecules comprisingfour polypeptide chains, two heavy (H) chains and two light (L) chainsinter-connected by disulfide bonds, as well as multimers thereof (e.g.,IgM). In a typical antibody, each heavy chain comprises a heavy chainvariable region (abbreviated herein as HCVR or V_(H)) and a heavy chainconstant region. The heavy chain constant region comprises threedomains, C_(H)1, C_(H)2 and C_(H)3. Each light chain comprises a lightchain variable region (abbreviated herein as LCVR or V_(L)) and a lightchain constant region. The light chain constant region comprises onedomain (C_(L)1). The V_(H) and V_(L) regions can be further subdividedinto regions of hypervariability, termed complementarity determiningregions (CDRs), interspersed with regions that are more conserved,termed framework regions (FR). Each V_(H) and V_(L) is composed of threeCDRs and four FRs, arranged from amino-terminus to carboxy-terminus inthe following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In differentembodiments of the invention, the FRs of the anti-IL-4R antibody (orantigen-binding portion thereof) may be identical to the human germlinesequences, or may be naturally or artificially modified. An amino acidconsensus sequence may be defined based on a side-by-side analysis oftwo or more CDRs.

The term “antibody,” as used herein, also includes antigen-bindingfragments of full antibody molecules. The terms “antigen-bindingportion” of an antibody, “antigen-binding fragment” of an antibody, andthe like, as used herein, include any naturally occurring, enzymaticallyobtainable, synthetic, or genetically engineered polypeptide orglycoprotein that specifically binds an antigen to form a complex.Antigen-binding fragments of an antibody may be derived, e.g., from fullantibody molecules using any suitable standard techniques such asproteolytic digestion or recombinant genetic engineering techniquesinvolving the manipulation and expression of DNA encoding antibodyvariable and optionally constant domains. Such DNA is known and/or isreadily available from, e.g., commercial sources, DNA libraries(including, e.g., phage-antibody libraries), or can be synthesized. TheDNA may be sequenced and manipulated chemically or by using molecularbiology techniques, for example, to arrange one or more variable and/orconstant domains into a suitable configuration, or to introduce codons,create cysteine residues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fabfragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fvfragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and(vii) minimal recognition units consisting of the amino acid residuesthat mimic the hypervariable region of an antibody (e.g., an isolatedcomplementarity determining region (CDR) such as a CDR3 peptide), or aconstrained FR3-CDR3-FR4 peptide. Other engineered molecules, such asdomain-specific antibodies, single domain antibodies, domain-deletedantibodies, chimeric antibodies, CDR-grafted antibodies, diabodies,triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalentnanobodies, bivalent nanobodies, etc.), small modularimmunopharmaceuticals (SMIPs), and shark variable IgNAR domains, arealso encompassed within the expression “antigen-binding fragment,” asused herein.

An antigen-binding fragment of an antibody will typically comprise atleast one variable domain. The variable domain may be of any size oramino acid composition and will generally comprise at least one CDRwhich is adjacent to or in frame with one or more framework sequences.In antigen-binding fragments having a V_(H) domain associated with aV_(L) domain, the V_(H) and V_(L) domains may be situated relative toone another in any suitable arrangement. For example, the variableregion may be dimeric and contain V_(H)-V_(H), V_(H)-V_(L) orV_(L)-V_(L) dimers. Alternatively, the antigen-binding fragment of anantibody may contain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibody maycontain at least one variable domain covalently linked to at least oneconstant domain. Non-limiting, exemplary configurations of variable andconstant domains that may be found within an antigen-binding fragment ofan antibody of the present invention include: (i) V_(H)-C_(H)1; (ii)V_(H)-C_(H)2; (iii) VH-CH3; (iv) V_(H)-C_(H)1-C_(H)2; (▾)V_(H)-C_(H)1-C_(H)2-C_(H)3; (vi) V_(H)-C_(H)2-C_(H)3; (vii) V_(H)-C_(L);V_(L)-C_(H)1; (ix) V_(L)-C_(H)2; (x) V_(L)-C_(H)3; (xi)V_(L)-C_(H)1-C_(H)2; (xii) V_(L)-C_(H)1-C_(H)2-C_(H)3;V_(L)-C_(H)2-C_(H)3; and (xiv) V_(L-)-C_(L). In any configuration ofvariable and constant domains, including any of the exemplaryconfigurations listed above, the variable and constant domains may beeither directly linked to one another or may be linked by a full orpartial hinge or linker region. A hinge region may consist of at least 2(e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in aflexible or semi-flexible linkage between adjacent variable and/orconstant domains in a single polypeptide molecule. Moreover, anantigen-binding fragment of an antibody of the present invention maycomprise a homo-dimer or hetero-dimer (or other multimer) of any of thevariable and constant domain configurations listed above in non-covalentassociation with one another and/or with one or more monomeric V_(H) orV_(L) domain (e.g., by disulfide bond(s)).

The term “antibody,” as used herein, also includes multispecific (e.g.,bispecific) antibodies. A multispecific antibody or antigen-bindingfragment of an antibody will typically comprise at least two differentvariable domains, wherein each variable domain is capable ofspecifically binding to a separate antigen or to a different epitope onthe same antigen. Any multispecific antibody format may be adapted foruse in the context of an antibody or antigen-binding fragment of anantibody of the present invention using routine techniques available inthe art. For example, the present invention includes methods comprisingthe use of bispecific antibodies wherein one arm of an immunoglobulin isspecific for PD-1 or a fragment thereof, and the other arm of theimmunoglobulin is specific for a second therapeutic target or isconjugated to a therapeutic moiety. Exemplary bispecific formats thatcan be used in the context of the present invention include, withoutlimitation, e.g., scFv-based or diabody bispecific formats, IgG-scFvfusions, dual variable domain (DVD)-Ig, Quadroma, knobs-into-holes,common light chain (e.g., common light chain with knobs-into-holes,etc.), CrossMab, CrossFab, (SEED) body, leucine zipper, Duobody,IgG1/IgG2, dual acting Fab (DAF)-IgG, and Mabe bispecific formats (see,e.g., Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein,for a review of the foregoing formats). Bispecific antibodies can alsobe constructed using peptide/nucleic acid conjugation, e.g., whereinunnatural amino acids with orthogonal chemical reactivity are used togenerate site-specific antibody-oligonucleotide conjugates which thenself-assemble into multimeric complexes with defined composition,valency and geometry. (See, e.g., Kazane et al., J. Am. Chem. Soc.[Epub: Dec. 4, 2012]).

The antibodies used in the methods of the present invention may be humanantibodies. The term “human antibody,” as used herein, is intended toinclude antibodies having variable and constant regions derived fromhuman germline immunoglobulin sequences. The human antibodies of theinvention may nonetheless include amino acid residues not encoded byhuman germline immunoglobulin sequences (e.g., mutations introduced byrandom or site-specific mutagenesis in vitro or by somatic mutation invivo), for example in the CDRs and in particular CDR3. However, the term“human antibody,” as used herein, is not intended to include antibodiesin which CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

The antibodies used in the methods of the present invention may berecombinant human antibodies. The term “recombinant human antibody,” asused herein, is intended to include all human antibodies that areprepared, expressed, created or isolated by recombinant means, such asantibodies expressed using a recombinant expression vector transfectedinto a host cell (described further below), antibodies isolated from arecombinant, combinatorial human antibody library (described furtherbelow), antibodies isolated from an animal (e.g., a mouse) that istransgenic for human immunoglobulin genes [see e.g., Taylor et al.(1992) Nucl. Acids Res. 20:6287-6295] or antibodies prepared, expressed,created or isolated by any other means that involves splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies have variable and constant regions derived from humangermline immunoglobulin sequences. In certain embodiments, however, suchrecombinant human antibodies are subjected to in vitro mutagenesis (or,when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the V_(H) andV_(L) regions of the recombinant antibodies are sequences that, whilederived from and related to human germline V_(H) and V_(L) sequences,may not naturally exist within the human antibody germline repertoire invivo.

According to certain embodiments, the antibodies used in the methods ofthe present invention specifically bind PD-1. The term “specificallybinds,” or the like, means that an antibody or antigen-binding fragmentthereof forms a complex with an antigen that is relatively stable underphysiologic conditions. Methods for determining whether an antibodyspecifically binds to an antigen are well known in the art and include,for example, equilibrium dialysis, surface plasmon resonance, and thelike. For example, an antibody that “specifically binds” PD-1, as usedin the context of the present invention, includes antibodies that bindPD-1 or portion thereof with a K_(D) of less than about 500 nM, lessthan about 300 nM, less than about 200 nM, less than about 100 nM, lessthan about 90 nM, less than about 80 nM, less than about 70 nM, lessthan about 60 nM, less than about 50 nM, less than about 40 nM, lessthan about 30 nM, less than about 20 nM, less than about 10 nM, lessthan about 5 nM, less than about 4 nM, less than about 3 nM, less thanabout 2 nM, less than about 1 nM or less than about 0.5 nM, as measuredin a surface plasmon resonance assay. An isolated antibody thatspecifically binds human PD-1 may, however, have cross-reactivity toother antigens, such as PD-1 molecules from other (non-human) species.

According to certain exemplary embodiments of the present invention, theanti-PD-1 antibody, or antigen-binding fragment thereof comprises aheavy chain variable region (HCVR), light chain variable region (LCVR),and/or complementarity determining regions (CDRs) comprising the aminoacid sequences of any of the anti-PD-1 antibodies as set forth in USPatent Publication No. 20150203579, hereby incorporated in its entirety.In certain exemplary embodiments, the anti-PD-1 antibody orantigen-binding fragment thereof that can be used in the context of themethods of the present invention comprises the heavy chaincomplementarity determining regions (HCDRs) of a heavy chain variableregion (HCVR) comprising the amino acid sequence of SEQ ID NO: 1 and thelight chain complementarity determining regions (LCDRs) of a light chainvariable region (LCVR) comprising the amino acid sequence of SEQ ID NO:2. According to certain embodiments, the anti-PD-1 antibody orantigen-binding fragment thereof comprises three HCDRs (HCDR1, HCDR2 andHCDR3) and three LCDRs (LCDR1, LCDR2 and LCDR3), wherein the HCDR1comprises the amino acid sequence of SEQ ID NO: 3; the HCDR2 comprisesthe amino acid sequence of SEQ ID NO: 4; the HCDR3 comprises the aminoacid sequence of SEQ ID NO: 5; the LCDR1 comprises the amino acidsequence of SEQ ID NO: 6; the LCDR2 comprises the amino acid sequence ofSEQ ID NO: 7; and the LCDR3 comprises the amino acid sequence of SEQ IDNO: 8. In yet other embodiments, the anti-PD-1 antibody orantigen-binding fragment thereof comprises an HCVR comprising SEQ ID NO:1 and an LCVR comprising SEQ ID NO: 2. In certain embodiments, themethods of the present invention comprise the use of an anti-PD-1antibody, wherein the antibody comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 9. In some embodiments, the anti-PD-1antibody comprises a light chain comprising the amino acid sequence ofSEQ ID NO: 10. An exemplary antibody comprising a heavy chain comprisingthe amino acid sequence of SEQ ID NO: 9 and a light chain comprising theamino acid sequence of SEQ ID NO: 10 is the fully human anti-PD-1antibody known as REGN2810 and also known as emplumab. According tocertain exemplary embodiments, the methods of the present inventioncomprise the use of REGN2810, or a bioequivalent thereof. The term“bioequivalent”, as used herein, refers to anti-PD-1 antibodies orPD-1-binding proteins or fragments thereof that are pharmaceuticalequivalents or pharmaceutical alternatives whose rate and/or extent ofabsorption do not show a significant difference with that of REGN2810when administered at the same molar dose under similar experimentalconditions, either single dose or multiple dose. In the context of theinvention, the term refers to antigen-binding proteins that bind to PD-1which do not have clinically meaningful differences with REGN2810 intheir safety, purity and/or potency.

According to certain embodiments of the present invention, theanti-human PD-1, or antigen-binding fragment thereof, comprises a HCVRhaving 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 1.

According to certain embodiments of the present invention, theanti-human PD-1, or antigen-binding fragment thereof, comprises a LCVRhaving 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 2.

According to certain embodiments of the present invention, theanti-human PD-1, or antigen-binding fragment thereof, comprises a HCVRcomprising an amino acid sequence of SEQ ID NO: 1 having no more than 5amino acid substitutions. According to certain embodiments of thepresent invention, the anti-human PD-1, or antigen-binding fragmentthereof, comprises a LCVR comprising an amino acid sequence of SEQ IDNO: 2 having no more than 2 amino acid substitutions.

Sequence identity may be measured by any method known in the art (e.g.,GAP, BESTFIT, and BLAST).

The present invention also includes use of anti-PD-1 antibodies inmethods to treat cancer, wherein the anti-PD-1 antibodies comprisevariants of any of the HCVR, LCVR and/or CDR amino acid sequencesdisclosed herein having one or more conservative amino acidsubstitutions. For example, the present invention includes use ofanti-PD-1 antibodies having HCVR, LCVR and/or CDR amino acid sequenceswith, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc.conservative amino acid substitutions relative to any of the HCVR, LCVRand/or CDR amino acid sequences disclosed herein.

Other anti-PD-1 antibodies that can be used in the context of themethods of the present invention include, e.g., the antibodies referredto and known in the art as nivolumab (US Patent No. 8008449),pembrolizumab (US Patent No. 8354509), MED10608 (US Patent No. 8609089),pidilizumab (US Patent No. 8686119), or any of the anti-PD-1 antibodiesas set forth in US Patent Nos. 6808710, 7488802, 8168757, 8354509,8779105, or 8900587.

The anti-PD-1 antibodies used in the context of the methods of thepresent invention may have pH-dependent binding characteristics. Forexample, an anti-PD-1 antibody for use in the methods of the presentinvention may exhibit reduced binding to PD-1 at acidic pH as comparedto neutral pH. Alternatively, an anti-PD-1 antibody of the invention mayexhibit enhanced binding to its antigen at acidic pH as compared toneutral pH. The expression “acidic pH” includes pH values less thanabout 6.2, e.g., about 6.0, 5.95, 5.9, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6,5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0, orless. As used herein, the expression “neutral pH” means a pH of about7.0 to about 7.4. The expression “neutral pH” includes pH values ofabout 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.

In certain instances, “reduced binding to PD-1 at acidic pH as comparedto neutral pH” is expressed in terms of a ratio of the K_(D) value ofthe antibody binding to PD-1 at acidic pH to the K_(D) value of theantibody binding to PD-1 at neutral pH (or vice versa). For example, anantibody or antigen-binding fragment thereof may be regarded asexhibiting “reduced binding to PD-1 at acidic pH as compared to neutralpH” for purposes of the present invention if the antibody orantigen-binding fragment thereof exhibits an acidic/neutral K_(D) ratioof about 3.0 or greater. In certain exemplary embodiments, theacidic/neutral K_(D) ratio for an antibody or antigen-binding fragmentof the present invention can be about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0,6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5,13.0, 13.5, 14.0, 14.5, 15.0, 20.0, 25.0, 30.0, 40.0, 50.0, 60.0, 70.0,100.0, or greater.

Antibodies with pH-dependent binding characteristics may be obtained,e.g., by screening a population of antibodies for reduced (or enhanced)binding to a particular antigen at acidic pH as compared to neutral pH.Additionally, modifications of the antigen-binding domain at the aminoacid level may yield antibodies with pH-dependent characteristics. Forexample, by substituting one or more amino acids of an antigen-bindingdomain (e.g., within a CDR) with a histidine residue, an antibody withreduced antigen-binding at acidic pH relative to neutral pH may beobtained. As used herein, the expression “acidic pH” means a pH of 6.0or less.

Combination Therapies

The methods of the present invention, according to certain embodiments,comprise administering to the subject a therapeutically effective amountof an anti-PD-1 antibody. In certain embodiments, the methods of thepresent invention comprise administering radiation therapy incombination with an anti-PD-1 antibody for additive or synergisticactivity to treat cancer. As used herein, the expression “in combinationwith” means that the radiation therapy is administered before, after, orconcurrent with the anti-PD-1 antibody. The term “in combination with”also includes sequential or concomitant administration of anti-PD-1antibody and radiation therapy. For example, when administered “before”the radiation therapy, the anti-PD-1 antibody may be administered morethan 150 hours, about 150 hours, about 100 hours, about 72 hours, about60 hours, about 48 hours, about 36 hours, about 24 hours, about 12hours, about 10 hours, about 8 hours, about 6 hours, about 4 hours,about 2 hours, about 1 hour, or about 30 minutes, about 15 minutes orabout 10 minutes prior to the administration of the radiation therapy.When administered “after” the radiation therapy, the anti-PD-1 antibodymay be administered about 10 minutes, about 15 minutes, about 30minutes, about 1 hour, about 2 hours, about 4 hours, about 6 hours,about 8 hours, about 10 hours, about 12 hours, about 24 hours, about 36hours, about 48 hours, about 60 hours, about 72 hours, or more than 72hours after the administration of the radiation therapy. Administration“concurrent” with the radiation therapy means that the anti-PD-1antibody is administered to the subject within less than 10 minutes(before, after, or at the same time) of administration of the radiationtherapy.

In certain embodiments, the methods of the present invention compriseadministration of an additional therapeutic agent wherein the additionaltherapeutic agent is an anti-cancer drug. As used herein, “anti-cancerdrug” means any agent useful to treat cancer including, but not limitedto, cytotoxins and agents such as antimetabolites, alkylating agents,anthracyclines, antibiotics, antimitotic agents, procarbazine,hydroxyurea, asparaginase, corticosteroids, mytotane (O, P′-(DDD)),biologics (e.g., antibodies and interferons) and radioactive agents. Asused herein, “a cytotoxin or cytotoxic agent”, also refers to achemotherapeutic agent and means any agent that is detrimental to cells.Examples include, but are not limited to, Taxol® (paclitaxel),temozolamide, cytochalasin B, gramicidin D, ethidium bromide, emetine,cisplatin, mitomycin, etoposide, tenoposide, vincristine, vinbiastine,coichicin, doxorubicin, daunorubicin, dihydroxy anthracin dione,mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin and analogs or homologs thereof.

In certain embodiments, the methods of the present invention compriseadministration of an additional therapeutic agent or therapeutic regimenor procedure selected from the group consisting of surgery, radiation, aprogrammed death ligand 1 (PD-L1) inhibitor (e.g., an anti-PD-L1antibody as disclosed in US Patent Publication 2015/0203580 oratezolizumab), a lymphocyte activation gene 3 (LAG-3) inhibitor (e.g.,an anti-LAG-3 antibody), a cytotoxic T-lymphocyte-associated protein 4(CTLA-4) inhibitor (e.g., ipilimumab), a glucocorticoid-induced tumornecrosis factor receptor (GITR) inhibitor (e.g., an anti-GITR antibody),a T-cell immunoglobulin and mucin containing -3 (TIM3) inhibitor, a B-and T-lymphocyte attenuator (BTLA) inhibitor, a T cell immunoreceptorwith Ig and ITIM domains (TIGIT) inhibitor, a CD47 inhibitor, anantagonist of another T-cell co-inhibitor or ligand (e.g., an antibodyto CD-28, 2B4, LY108, LAIR1, ICOS, CD160 or VISTA), a CD20 inhibitor(e.g., an anti-CD20 antibody, or a bispecific CD3/CD20 antibody) anindoleamine-2,3-dioxygenase (IDO) inhibitor, a vascular endothelialgrowth factor (VEGF) antagonist [e.g., a “VEGF-Trap” such as afliberceptor other VEGF-inhibiting fusion protein as set forth in US 7,087,411, oran anti-VEGF antibody or antigen binding fragment thereof (e.g.,bevacizumab, or ranibizumab) or a small molecule kinase inhibitor ofVEGF receptor (e.g., sunitinib, sorafenib, or pazopanib)], anangiopoietin 2 (Ang2) inhibitor (e.g., nesvacumab), a transforminggrowth factor beta (TGFβ) inhibitor, a CD38 inhibitor, an epidermalgrowth factor receptor (EGFR) inhibitor (e.g., erlotinib, cetuximab), anagonist to a co-stimulatory receptor (e.g., an agonist toglucocorticoid-induced TNFR-related protein), an antibody to atumor-specific antigen [e.g., CA9, CA125, melanoma-associated antigen 3(MAGE3), carcinoembryonic antigen (CEA), vimentin, tumor-M2-PK,prostate-specific antigen (PSA), mucin-1, MART-1, and CA19-9], a vaccine(e.g., Bacillus Calmette-Guerin, a cancer vaccine), cyclophosphamide, anadjuvant to increase antigen presentation (e.g., granulocyte macrophagecolony-stimulating factor), a cytotoxin, a chemotherapeutic agent (e.g.,dacarbazine, temozolomide, docetaxel, doxorubicin, daunorubicin,cisplatin, carboplatin, gemcitabine, methotrexate, mitoxantrone,oxaliplatin, paclitaxel, and vincristine), an interleukin-6 receptor(IL-6R) inhibitor (e.g., sarilumab), an IL-4R inhibitor (e.g.,dupilumab), an IL-10 inhibitor, a cytokine such as IL-2, IL-7, IL-21,and IL-15, an antibody-drug conjugate (ADC) (e.g., anti-CD19-DM4 ADC,and anti-DS6-DM4 ADC), chimeric antigen receptor T cells (e.g.,CD19-targeted T cells), an anti-inflammatory drug (e.g.,corticosteroids, and non-steroidal anti-inflammatory drugs), and adietary supplement such as anti-oxidants.

In certain embodiments, the methods of the invention compriseadministering an anti-PD-1 antibody in combination with radiationtherapy and optionally, an anti-GITR antibody to generate long-termdurable anti-tumor responses and/or enhance survival of patients withcancer. In some embodiments, the methods of the invention compriseadministering radiation therapy prior to, concomitantly or afteradministering an anti-PD-1 antibody and an anti-GITR antibody to acancer patient. For example, radiation therapy may be administered inone or more doses to tumor lesions after administration of one or moredoses of the antibodies. In some embodiments, radiation therapy may beadministered locally to a tumor lesion to enhance the localimmunogenicity of a patient's tumor (adjuvinating radiation) and/or tokill tumor cells (ablative radiation) after systemic administration ofan anti-PD-1 antibody and/or an anti-GITR antibody. In certainembodiments, the radiation therapy is administered to a first tumorlesion, but not to a second tumor lesion, wherein the administration incombination with the anti-PD-1 antibody leads to tumor regression inboth the first and second tumor lesions (abscopal effect). In certainembodiments, the methods of the present invention comprise administeringan anti-PD-1 antibody in combination with radiation therapy andoptionally, an anti-GITR antibody to generate prolonged abscopal effect.

In certain embodiments, an anti-PD-1 antibody may be administered incombination with radiation therapy and a chemotherapeutic agent (e.g.,temozolomide or cyclophosphamide), a VEGF antagonist (e.g.,aflibercept), or granulocyte macrophage colony-stimulating factor.

Pharmaceutical Compositions and Administration

The present invention includes methods which comprise administering ananti-PD-1 antibody in combination with radiation to a subject whereinthe anti-PD-1 antibody is contained within a pharmaceutical composition.The pharmaceutical compositions of the invention may be formulated withsuitable carriers, excipients, and other agents that provide suitabletransfer, delivery, tolerance, and the like. A multitude of appropriateformulations can be found in the formulary known to all pharmaceuticalchemists: Remington's Pharmaceutical Sciences, Mack Publishing Company,Easton, PA. These formulations include, for example, powders, pastes,ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)containing vesicles (such as LIPOFECTINTM), DNA conjugates, anhydrousabsorption pastes, oil-in-water and water-in-oil emulsions, emulsionscarbowax (polyethylene glycols of various molecular weights), semi-solidgels, and semi-solid mixtures containing carbowax. See also Powell etal. “Compendium of excipients for parenteral formulations” PDA (1998) JPharm Sci Technol 52:238-311.

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the mutant viruses, receptor mediated endocytosis (see, e.g.,Wu et al., 1987, J. Biol. Chem. 262: 4429-4432). Methods ofadministration include, but are not limited to, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, and oral routes. The composition may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents.

A pharmaceutical composition of the present invention can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present invention. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

In certain situations, the pharmaceutical composition can be deliveredin a controlled release system. In one embodiment, a pump may be used.In another embodiment, polymeric materials can be used; see, MedicalApplications of Controlled Release, Langer and Wise (eds.), 1974, CRCPres., Boca Raton, Florida. In yet another embodiment, a controlledrelease system can be placed in proximity of the composition's target,thus requiring only a fraction of the systemic dose (see, e.g., Goodson,1984, in Medical Applications of Controlled Release, supra, vol. 2, pp.115-138). Other controlled release systems are discussed in the reviewby Langer, 1990, Science 249:1527-1533.

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous and intramuscular injections, dripinfusions, etc. These injectable preparations may be prepared by knownmethods. For example, the injectable preparations may be prepared, e.g.,by dissolving, suspending or emulsifying the antibody or its saltdescribed above in a sterile aqueous medium or an oily mediumconventionally used for injections. As the aqueous medium forinjections, there are, for example, physiological saline, an isotonicsolution containing glucose and other auxiliary agents, etc., which maybe used in combination with an appropriate solubilizing agent such as analcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80,HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)],etc. As the oily medium, there are employed, e.g., sesame oil, soybeanoil, etc., which may be used in combination with a solubilizing agentsuch as benzyl benzoate, benzyl alcohol, etc. The injection thusprepared is preferably filled in an appropriate ampoule.

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc.

In certain embodiments, the present invention provides a pharmaceuticalformulation comprising a therapeutic amount of an anti-PD-1 antibody anda pharmaceutical carrier. In certain embodiments, the present inventionprovides for an anti-PD-1 antibody formulated in a pharmaceuticalcomposition for use in intravenous administration.

Administration Regimens

The present invention includes methods comprising administering to asubject an anti-PD-1 antibody at a dosing frequency of about four timesa week, twice a week, once a week, once every two weeks, once everythree weeks, once every four weeks, once every five weeks, once everysix weeks, once every eight weeks, once every twelve weeks, or lessfrequently so long as a therapeutic response is achieved. In certainembodiments, the present invention includes methods comprisingadministering to a subject radiation therapy at a dosing frequency ofabout seven times a week, about four times a week, twice a week, once aweek, once every two weeks, once every three weeks, once every fourweeks, once every five weeks, once every six weeks, once every eightweeks, once every twelve weeks, or less frequently so long as atherapeutic response is achieved. In certain embodiments, the methodsinvolve the administration of an anti-PD-1 antibody in combination withradiation therapy at a dosing frequency of about seven times a week,about four times a week, twice a week, once a week, once every twoweeks, once every three weeks, once every four weeks, once every fiveweeks, once every six weeks, once every eight weeks, once every nineweeks, once every twelve weeks, or less frequently so long as atherapeutic response is achieved.

In certain embodiments, the methods of the present invention compriseadministering radiation therapy wherein the radiation therapy ishypofractionated radiation therapy. In certain embodiments, thehypofractionated radiation therapy comprises 2-12 fractions. In certainembodiments, the 2-12 fractions are administered on consecutive days. Incertain embodiments, the radiation therapy is administered afteradministering one or more doses of an anti-PD-1 antibody. In certainembodiments, the anti-PD-1 antibody is administered 0.5-2 weeks beforeadministration of one or more fractions of radiation therapy.

According to certain embodiments of the present invention, multipledoses of an anti-PD-1 antibody in combination with radiation therapy maybe administered to a subject over a defined time course. The methodsaccording to this aspect of the invention comprise sequentiallyadministering to a subject one or more doses of an anti-PD-1 antibody incombination with one or more doses of radiation. As used herein,“sequentially administering” means that each dose of the antibody isadministered to the subject at a different point in time, e.g., ondifferent days separated by a predetermined interval (e.g., hours, days,weeks or months). In certain embodiments, the methods of the presentinvention comprise sequentially administering one or more doses of ananti-PD-1 antibody wherein each dose is administered 0.5-12 weeks afterthe immediately preceding dose. In certain further embodiments, themethods further comprise administering radiation therapy. The radiationtherapy may be hypofractionated radiation therapy. In certainembodiments, the radiation therapy comprises 2-12 fractions. In someembodiments, the radiation fractions are administered on consecutivedays or alternate days. In certain embodiments, the radiation fractionsare administered once in 3 days, once in 4 days, once in 5 days, once in6 days, once in 7 days, or a combination thereof.

In certain embodiments, the present invention includes methods whichcomprise sequentially administering to the patient a single initial doseof an anti-PD-1 antibody, followed by one or more secondary doses of theanti-PD-1 antibody, and optionally followed by one or more tertiarydoses of the anti-PD-1 antibody. In certain embodiments, the methodsfurther comprise sequentially administering to the patient a singleinitial dose of radiation therapy, followed by one or more secondarydoses of radiation therapy, and optionally followed by one or moretertiary doses of the radiation therapy. In alternate embodiments, themethods further comprise sequentially administering one or morefractions of hypofractionated radiation therapy.

According to certain embodiments of the present invention, multipledoses of an anti-PD-1 antibody and radiation therapy may be administeredto a subject over a defined time course. The methods according to thisaspect of the invention comprise sequentially administering to a subjectmultiple doses of an anti-PD-1 antibody and radiation. As used herein,“sequentially administering” means that each dose of the anti-PD-1antibody in combination with the radiation therapy is administered tothe subject at a different point in time, e.g., on different daysseparated by a predetermined interval (e.g., hours, days, weeks ormonths).

The terms “initial dose,” “secondary doses,” and “tertiary doses,” referto the temporal sequence of administration. Thus, the “initial dose” isthe dose which is administered at the beginning of the treatment regimen(also referred to as the “baseline dose”); the “secondary doses” are thedoses which are administered after the initial dose; and the “tertiarydoses” are the doses which are administered after the secondary doses.The initial, secondary, and tertiary doses may all contain the sameamount of the antibody (anti-PD-1 antibody). In certain embodiments,however, the amount contained in the initial, secondary and/or tertiarydoses varies from one another (e.g., adjusted up or down as appropriate)during the course of treatment. In certain embodiments, one or more(e.g., 1, 2, 3, 4, or 5) doses are administered at the beginning of thetreatment regimen as “loading doses” followed by subsequent doses thatare administered on a less frequent basis (e.g., “maintenance doses”).For example, an anti-PD-1 antibody may be administered to a patient witha cancer at a loading dose of about 1-3 mg/kg followed by one or moremaintenance doses of about 0.1 to about 20 mg/kg of the patient's bodyweight.

In one exemplary embodiment of the present invention, each secondaryand/or tertiary dose is administered % to 14 (e.g., ¹/₂, 1, 1%, 2, 2%,3, 3%, 4, 4%, 5, 5%, 6, 6%, 7, 7%, 8, 8%, 9, 9%, 10, 10%, 11, 11%, 12,12%, 13, 13%, 14, 14%, or more) weeks after the immediately precedingdose. The phrase “the immediately preceding dose,” as used herein,means, in a sequence of multiple administrations, the dose of anti-PD-1antibody (and/or radiation) which is administered to a patient prior tothe administration of the very next dose in the sequence with nointervening doses.

The methods according to this aspect of the invention may compriseadministering to a patient any number of secondary and/or tertiary dosesof an anti-PD-1 antibody (and/or radiation therapy). For example, incertain embodiments, only a single secondary dose is administered to thepatient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8,or more) secondary doses are administered to the patient. Likewise, incertain embodiments, only a single tertiary dose is administered to thepatient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8,or more) tertiary doses are administered to the patient.

In embodiments involving multiple secondary doses, each secondary dosemay be administered at the same frequency as the other secondary doses.For example, each secondary dose may be administered to the patient 1 to2 weeks after the immediately preceding dose. Similarly, in embodimentsinvolving multiple tertiary doses, each tertiary dose may beadministered at the same frequency as the other tertiary doses. Forexample, each tertiary dose may be administered to the patient 2 to 4weeks after the immediately preceding dose. Alternatively, the frequencyat which the secondary and/or tertiary doses are administered to apatient can vary over the course of the treatment regimen. The frequencyof administration may also be adjusted during the course of treatment bya physician depending on the needs of the individual patient followingclinical examination.

In certain embodiments, one or more doses of an anti-PD-1 antibodyand/or radiation are administered at the beginning of a treatmentregimen as “induction doses” on a more frequent basis (twice a week,once a week or once in 2 weeks) followed by subsequent doses(“consolidation doses” or “maintenance doses”) that are administered ona less frequent basis (e.g., once in 2-12 weeks). In certainembodiments, one or more doses of an anti-PD-1 antibody and/or radiationare administered at the beginning of a treatment regimen as “inductiondoses” on a more frequent basis (twice a week, once a week or once in 2weeks) followed by subsequent doses of the anti-PD-1 antibody.

The present invention includes methods which comprise sequentiallyadministering one or more doses of an anti-PD-1 antibody in combinationwith one or more doses of radiation therapy wherein the one or moredoses are comprised in one or more treatment cycles.

According to certain embodiments of the present invention, the methodscomprise administering at least one treatment cycle wherein the at leastone treatment cycle comprises administration of one or more doses of ananti-PD-1 antibody, and optionally one or more doses of radiationtherapy. In certain embodiments, a treatment cycle comprises 1-10 dosesof the anti-PD-1 antibody wherein each dose of the anti-PD-1 antibody isadministered 0.5-8 weeks after the immediately preceding dose. Incertain embodiments, the methods of the present invention compriseadministration of up to 6 or 8 treatment cycles. In certain otherembodiments, the methods of the present invention compriseadministration of up to 12 treatment cycles, or more as required fortherapeutic effect. In certain embodiments, at least one treatment cyclefurther comprises radiation therapy. In some embodiments, the radiationtherapy is hypofractionated radiation therapy, wherein thehypofractionated radiation therapy comprises 2-12 fractions. In certainembodiments, the 2-12 fractions are administered on consecutive days.

The present invention includes methods comprising sequentialadministration of an anti-PD-1 antibody in combination with radiationtherapy, to a patient to treat a cancer (e.g., a solid tumor). In someembodiments, the present methods comprise administering one or moredoses of an anti-PD-1 antibody followed by radiation therapy. In certainfurther embodiments, the radiation therapy is administered in fractions(hypofractionated radiation). In certain embodiments, the presentmethods comprise administering a single dose of an anti-PD-1 antibodyfollowed by 2-10 fractions of radiation therapy followed by one or moredoses of the anti-PD-1 antibody. In some embodiments, one or more dosesof about 0.1 mg/kg to about 20 mg/kg of an anti-PD-1 antibody may beadministered followed by radiation therapy to inhibit tumor growthand/or to prevent tumor recurrence in a subject with a cancer (e.g., asolid tumor). In some embodiments, the anti-PD-1 antibody isadministered at one or more doses followed by radiation therapyresulting in increased anti-tumor efficacy (e.g., greater inhibition oftumor growth, increased prevention of tumor recurrence as compared to anuntreated subject or a subject administered with either antibody orradiation as monotherapy). Alternative embodiments of the inventionpertain to concomitant administration of anti-PD-1 antibody andradiation which is administered at a similar or different frequencyrelative to the anti-PD-1 antibody. In some embodiments, the radiationtherapy is administered before, after or concurrently with the anti-PD-1antibody.

Dosage

The amount of anti-PD-1 antibody administered to a subject according tothe methods of the present invention is, generally, a therapeuticallyeffective amount. As used herein, the phrase “therapeutically effectiveamount” means an amount of antibody (anti-PD-1 antibody that results inone or more of: (a) a reduction in the severity or duration of a symptomor an indication of a cancer, e.g., a solid tumor; (b) inhibition oftumor growth, or an increase in tumor necrosis, tumor shrinkage and/ortumor disappearance; (c) delay in tumor growth and development; (d)inhibition of tumor metastasis; (e) prevention of recurrence of tumorgrowth; (f) increase in survival of a subject with a cancer; and/or (g)a reduction in the use or need for conventional anti-cancer therapy(e.g., reduced or eliminated use of chemotherapeutic or cytotoxicagents) as compared to an untreated subject or a subject administeredwith the antibody as monotherapy.

In the case of an anti-PD-1 antibody, a therapeutically effective amountcan be from about 0.05 mg to about 600 mg, from about 1 mg to about 500mg, from about 10 mg to about 450 mg, from about 50 mg to about 400 mg,from about 75 mg to about 350 mg, or from about 100 mg to about 300 mgof the antibody. For example, in various embodiments, the amount of theanti-PD-1 antibody is about 0.05 mg, about 0.1 mg, about 1.0 mg, about1.5 mg, about 2.0 mg, about 10 mg, about 20 mg, about 30 mg, about 40mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg,about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg,about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg,about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg,about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg,about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg,about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg,about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg,about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg,about 500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg,about 550 mg, about 560 mg, about 570 mg, about 580 mg, about 590 mg, orabout 600 mg, of the anti-PD-1 antibody. In one embodiment, 250 mg of ananti-PD-1 antibody is administered according to the methods of thepresent invention. In one embodiment, 200 mg of an anti-PD-1 antibody isadministered according to the methods of the present invention. . In oneembodiment, 350 mg of an anti-PD-1 antibody is administered according tothe methods of the present invention.

The amount of either anti-PD-1 antibody contained within the individualdoses may be expressed in terms of milligrams of antibody per kilogramof subject body weight (i.e., mg/kg). In certain embodiments, theanti-PD-1 antibody used in the methods of the present invention may beadministered to a subject at a dose of about 0.0001 to about 100 mg/kgof subject body weight. In certain embodiments, an anti-PD-1 antibodymay be administered at dose of about 0.1 mg/kg to about 20 mg/kg of apatient's body weight. In certain embodiments, the methods of thepresent invention comprise administration of an anti-PD-1 antibody at adose of about 1 mg/kg, 3 mg/kg, 5 mg/kg or 10 mg/kg of a patient's bodyweight.

In certain embodiments, the amount of anti-PD-1 antibody administered toa patient may be less than a therapeutically effective amount, i.e., asubtherapeutic dose. For example, if the therapeutically effectiveamount of an anti-PD-1 antibody comprises 3 mg/kg, a subtherapeutic dosecomprises an amount less than 3 mg/kg, e.g., 2 mg/kg, 1.5 mg/kg, 1mg/kg, 0.5 mg/kg or 0.3 mg/kg. As defined herein, a “subtherapeuticdose” refers to an amount of the anti-PD-1 antibody that does not leadto a therapeutic effect by itself. However, in certain embodiments, asubtherapeutic dose of an anti-PD-1 antibody is administered with asecond and optionally a third therapeutic agent to promote a therapeuticeffect.

In certain embodiments, the radiation therapy administered to a subjectin need thereof comprises 2-100 Gray (Gy). In certain embodiments, theradiation therapy comprises 5, 7, 8, 9, 10, 11, 12, 15, 20, 23, 25, 27,30, 35, 40, or 45 Gy. In certain other embodiments, the radiationtherapy comprises 50-100, 60-90, or 70-80 Gy. In certain embodiments,the radiation therapy is administered in 2-12 fractions(hypofractionated radiation therapy), wherein each fraction comprises2-10 Gy. For example, 30 Gy of radiation is administered comprised in 5fractions, each fraction comprising 6 Gy.

Selected Embodiments

Selected embodiments of the present invention include the following:

In some embodiments, the present disclosure provides a method oftreating or inhibiting the growth of a tumor in a subject comprising:

(a) selecting a subject with a cancer; and

(b) administering to the subject in need thereof one or more doses ofradiation therapy in combination with one or more doses of atherapeutically effective amount of an antibody or antigen-bindingfragment thereof that specifically binds programmed death 1 (PD-1)wherein the administration of the combination results in enhancedtherapeutic efficacy as compared to administration of the antibody orradiation alone.

In one embodiment, each dose of the anti-PD-1 antibody comprises between0.1-20 mg/kg of the subject's body weight.

In another embodiment, each dose of the anti-PD-1 antibody comprises0.3, 1, 3, 5 or 10 mg/kg of the subject's body weight.

In other embodiments, each dose of the anti-PD-1 antibody comprises20-400 mg.

In some embodiments, each dose of the anti-PD-1 antibody comprises 200mg.

In one embodiment, each dose of radiation comprises 2-80 Gray (Gy).

In another embodiment, each dose of the anti-PD-1 antibody comprises 1,3, or 10 mg/kg of the subject's body weight and each dose of radiationtherapy comprises 20-50 Gy.

In other embodiments, the radiation therapy is fractionated radiationtherapy.

In some embodiments, the fractionated radiation therapy comprises 2-10fractions.

In one embodiment, the fractionated radiation therapy comprises 30 Gy in5 fractions.

In another embodiment, the fractionated radiation therapy comprises 27Gy in 3 fractions.

In other embodiments, 4-50 doses of the anti-PD-1 antibody areadministered, and wherein each dose is administered 0.5-4 weeks afterthe immediately preceding dose.

In some embodiments, each dose of the anti-PD-1 antibody is administered2 weeks after the immediately preceding dose.

In one embodiment, the anti-PD-1 antibody is administered prior to,concurrent with or after the radiation therapy.

In another embodiment, the anti-PD-1 antibody is administered prior tothe radiation therapy.

In other embodiments, the anti-PD-1 antibody is administered 1 weekprior to radiation therapy.

In some embodiments, enhanced therapeutic efficacy comprises an effectselected from the group consisting of tumor regression, abscopal effect,inhibition of tumor metastasis, reduction in metastatic lesions overtime, reduced use of chemotherapeutic or cytotoxic agents, reduction intumor burden, increase in progression-free survival, increase in overallsurvival, complete response, partial response, and stable disease.

In one embodiment, enhanced therapeutic efficacy comprises tumorregression in a tumor distal to an irradiated tumor.

In another embodiment, the tumor growth is inhibited by at least 50% ascompared to a subject administered with either antibody or radiationalone.

In other embodiments, the tumor growth is inhibited by at least 50% ascompared to a subject administered a dose of radiation prior to ananti-PD-1 antibody.

In some embodiments, the present disclosure provides a method oftreating a tumor comprising: (a) selecting a subject with a cancer; and(b) administering to the subject at least one treatment cycle whereinthe at least one treatment cycle comprises 1-6 doses of an anti-PD-1antibody and wherein each dose is administered 2 weeks after theimmediately preceding dose.

In one embodiment, each dose of the anti-PD-1 antibody comprises 1, 3, 5or 10 mg/kg of the subject's body weight.

In another embodiment, the at least one treatment cycle furthercomprises radiation therapy.

In other embodiments, the radiation therapy comprises about 20-50 Gy.

In some embodiments, the radiation therapy comprises about 27 Gy.

In one embodiment, the radiation therapy comprises about 30 Gy.

In another embodiment, the radiation therapy is fractionated radiationtherapy.

In other embodiments, the fractionated radiation therapy comprises 2-6fractions.

In some embodiments, the fractionated radiation therapy comprises 3fractions.

In one embodiment, the fractionated radiation therapy comprises 5fractions.

In another embodiment, the radiation therapy comprises about 27 Gy in 3fractions.

In other embodiments, the radiation therapy comprises about 30 Gy in 5fractions.

In some embodiments, the fractions are administered on sequential days.

In one embodiment, the anti-PD-1 antibody is administered 1 week beforeradiation therapy.

In another embodiment, up to 10 treatment cycles are administered to thesubject in need thereof.

In other embodiments, 6 treatment cycles are administered to the subjectin need thereof.

In some embodiments, radiation therapy is administered in the 1sttreatment cycle.

In one embodiment, the radiation therapy comprises about 20-50 Gy.

In another embodiment, the radiation therapy comprises hypofractionatedradiation therapy.

In other embodiments, the fractionated radiation therapy comprises 2-6fractions.

In some embodiments, the radiation therapy comprises about 27 Gy in 3fractions.

In one embodiment, the radiation therapy comprises about 30 Gy in 5fractions.

In another embodiment, the fractions are administered on sequentialdays.

In other embodiments, the anti-PD-1 antibody is administered 1 weekbefore radiation therapy.

In some embodiments, each treatment cycle comprises 4 doses of theanti-PD-1 antibody.

In one embodiment, the treatment produces a therapeutic effect selectedfrom the group consisting of inhibition of tumor growth, tumorregression, reduction in the size of a tumor, reduction in tumor cellnumber, delay in tumor growth, abscopal effect, inhibition of tumormetastasis, reduction in metastatic lesions over time, reduced use ofchemotherapeutic or cytotoxic agents, reduction in tumor burden,increase in progression-free survival, increase in overall survival,complete response, partial response, and stable disease.

In another embodiment, the treatment effect comprises tumor regressionin a tumor distal to an irradiated tumor in the subject.

In other embodiments, tumor growth is delayed by at least 10 days ascompared to an untreated subject.

In some embodiments, the tumor growth is inhibited by at least 50% ascompared to an untreated subject.

In one embodiment, the tumor growth is inhibited by at least 50% ascompared to a subject administered with either antibody or radiationalone.

In some embodiments, the present disclosure provides a method oftreating a tumor comprising: (a) selecting a subject with a first solidtumor lesion and a second solid tumor lesion, wherein the second solidtumor lesion is located distally from the first solid tumor lesion; and(b) administering an anti-PD-1 antibody or antigen-binding fragmentthereof in combination with radiation therapy.

In other embodiments, the radiation therapy is administered to the firsttumor lesion but not the second tumor lesion and wherein theadministration leads to tumor regression in both the first and secondtumor lesions.

In one embodiment, the anti-PD-1 antibody is administered beforeradiation therapy.

In another embodiment, the subject is resistant or inadequatelyresponsive to, or relapsed after prior therapy.

In other embodiments, the cancer is recurrent or metastatic cancer.

In some embodiments, the method further comprising administering to thesubject an additional therapeutic agent or therapy, wherein theadditional therapeutic agent or therapy is selected from the groupconsisting of surgery, a chemotherapeutic agent, a cancer vaccine, aprogrammed death ligand 1 (PD-L1) inhibitor, a lymphocyte activationgene 3 (LAG3) inhibitor, a cytotoxic T-lymphocyte-associated protein 4(CTLA-4) inhibitor, a glucocorticoid-induced tumor necrosis factorreceptor (GITR) inhibitor, a T-cell immunoglobulin and mucin-domaincontaining-3 (TIM3) inhibitor, a B- and T-lymphocyte attenuator (BTLA)inhibitor, a T cell immunoreceptor with Ig and ITIM domains (TIGIT)inhibitor, a CD47 inhibitor, an indoleamine-2,3-dioxygenase (IDO)inhibitor, a bispecific anti-CD3/anti-CD20 antibody, a vascularendothelial growth factor (VEGF) antagonist, an angiopoietin-2 (Ang2)inhibitor, a transforming growth factor beta (TGFβ) inhibitor, anepidermal growth factor receptor (EGFR) inhibitor,granulocyte-macrophage colony-stimulating factor (GM-CSF),cyclophosphamide, an antibody to a tumor-specific antigen, BacillusCalmette-Guerin vaccine, a cytotoxin, an interleukin 6 receptor (IL-6R)inhibitor, an interleukin 4 receptor (IL-4R) inhibitor, an IL-10inhibitor, IL-2, IL-7, IL-21, IL-15, an antibody-drug conjugate, ananti-inflammatory drug, and a dietary supplement.

In one embodiment, the additional therapeutic agent is an anti-GITRantibody.

In another embodiment, the additional therapeutic agent iscyclophosphamide.

In other embodiments, the additional therapeutic agent is GM-CSF.

In some embodiments, the additional therapeutic agent is selected fromthe group consisting of docetaxel, carboplatin, paclitaxel, cisplatin,gemcitabine, and pemetrexed.

In one embodiment, the anti-PD-1 antibody is administered intravenously,subcutaneously, or intraperitoneally.

In another embodiment, the cancer comprises a solid tumor.

In other embodiments, the solid tumor is selected from the groupconsisting of colorectal cancer, ovarian cancer, prostate cancer, breastcancer, brain cancer, cervical cancer, bladder cancer, anal cancer,uterine cancer, colon cancer, liver cancer, pancreatic cancer, lungcancer, endometrial cancer, bone cancer, testicular cancer, skin cancer,kidney cancer, stomach cancer, esophageal cancer, head and neck cancer,salivary gland cancer, and myeloma.

In some embodiments, the solid tumor is selected from the groupconsisting of hepatocellular carcinoma, non-small cell lung cancer, headand neck squamous cell cancer, basal cell carcinoma, breast carcinoma,cutaneous squamous cell carcinoma, chondrosarcoma, angiosarcoma,cholangiocarcinoma, soft tissue sarcoma, colorectal cancer, melanoma,Merkel cell carcinoma, and glioblastoma multiforme.

In one embodiment, the anti-PD-1 antibody or antigen-binding fragmentthereof comprises the heavy chain complementarity determining regions(HCDR1, HCDR2 and HCDR3) of a heavy chain variable region (HCVR)comprising the amino acid sequence of SEQ ID NO: 1 and three light chaincomplementarity determining regions (LCDR1, LCDR2 and LCDR3) of a lightchain variable region (LCVR) comprising the amino acid sequence of SEQID NO: 2.

In another embodiment, the anti-PD-1 antibody or antigen-bindingfragment thereof comprises three HCDRs (HCDR1, HCDR2 and HCDR3) andthree LCDRs (LCDR1, LCDR2 and LCDR3), wherein HCDR1 comprises the aminoacid sequence of SEQ ID NO: 3; HCDR2 comprises the amino acid sequenceof SEQ ID NO: 4; HCDR3 comprises the amino acid sequence of SEQ ID NO:5; LCDR1 comprises the amino acid sequence of SEQ ID NO: 6; LCDR2comprises the amino acid sequence of SEQ ID NO: 7; and LCDR3 comprisesthe amino acid sequence of SEQ ID NO: 8.

In other embodiments, the HCVR comprises the amino acid sequence of SEQID NO: 1 and the LCVR comprises the amino acid sequence of SEQ ID NO: 2.

In another embodiment, the anti-PD-1 antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 9 and a light chaincomprising the amino acid sequence of SEQ ID NO: 10.

In some embodiments, the present disclosure provides a method forinhibiting the activation and/or proliferation of T regulatory (Treg)cells comprising: (a) selecting a subject with a solid tumor; and (b)administering to the subject (i) an anti-PD-1 antibody orantigen-binding fragment thereof, (ii) radiation therapy and (iii) atleast one of an antibody or antigen-binding fragment thereof that bindsspecifically to glucocorticoid-induced tumor necrosis factor receptor(GITR), cyclophosphamide, GM-CSF, an anti-LAG3 antibody, docetaxel, orcarboplatin.

In one embodiment, the subject has a large tumor.

In another embodiment, the radiation dose is 2-50 Gy.

In other embodiments, the administration leads to at least one effectselected from the group consisting of inhibition of tumor growth, tumorregression, reduction in the size of a tumor, reduction in tumor cellnumber, delay in tumor growth, abscopal effect, inhibition of tumormetastasis, reduction in metastatic lesions over time, reduced use ofchemotherapeutic or cytotoxic agents, reduction in tumor burden,increase in progression-free survival, increase in overall survival,complete response, partial response, and stable disease.

In some embodiments, the solid tumor is selected from the groupconsisting of colorectal cancer, ovarian cancer, prostate cancer, breastcancer, brain cancer, cervical cancer, bladder cancer, anal cancer,uterine cancer, colon cancer, liver cancer, pancreatic cancer, lungcancer, endometrial cancer, bone cancer, testicular cancer, skin cancer,kidney cancer, stomach cancer, esophageal cancer, head and neck cancer,salivary gland cancer, and myeloma.

In one embodiment, the anti-PD-1 antibody or antigen-binding fragmentthereof comprises the heavy chain complementarity determining regions(HCDR1, HCDR2 and HCDR3) of a heavy chain variable region (HCVR)comprising the amino acid sequence of SEQ ID NO: 1 and three light chaincomplementarity determining regions (LCDR1, LCDR2 and LCDR3) of a lightchain variable region (LCVR) comprising the amino acid sequence of SEQID NO: 2.

In another embodiment, the anti-PD-1 antibody or antigen-bindingfragment thereof comprises three HCDRs (HCDR1, HCDR2 and HCDR3) andthree LCDRs (LCDR1, LCDR2 and LCDR3), wherein HCDR1 comprises the aminoacid sequence of SEQ ID NO: 3; HCDR2 comprises the amino acid sequenceof SEQ ID NO: 4; HCDR3 comprises the amino acid sequence of SEQ ID NO:5; LCDR1 comprises the amino acid sequence of SEQ ID NO: 6; LCDR2comprises the amino acid sequence of SEQ ID NO: 7; and LCDR3 comprisesthe amino acid sequence of SEQ ID NO: 8.

In other embodiments, the HCVR comprises the amino acid sequence of SEQID NO: 1 and the LCVR comprises the amino acid sequence of SEQ ID NO: 2.

In one embodiment, the anti-PD-1 antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 9 and a light chaincomprising the amino acid sequence of SEQ ID NO: 10.

In some embodiments, the present disclosure provides a method oftreating or inhibiting the growth of a tumor comprising: (a) selecting asubject with a skin cancer; and (b) administering to the subject in needthereof a therapeutically effective amount of an antibody orantigen-binding fragment thereof that specifically binds PD-1.

In one embodiment, said antibody or antigen-binding fragment thereofthat specifically binds PD-1 is administered as a monotherapy.

In another embodiment, said skin cancer is an UV-associated skin cancer.

In other embodiments, said skin cancer is selected from the groupconsisting of cutaneous squamous cell carcinoma (CSCC), basal cellcarcinoma (BCC), Merkel cell carcinoma and melanoma.

In some embodiments, with the proviso that said skin cancer is not asquamous cell carcinoma of head and neck.

In one embodiment, said skin cancer is a metastatic, unresectable and/orlocally advanced cancer.

In another embodiment, said skin cancer is BCC, and wherein said patientis intolerant to or progresses after treatment with a hedgehog pathwayinhibitor.

In other embodiments, said antibody or antigen-binding fragment thereofis administered as one or more doses, wherein each dose is administered0.5 to 4 weeks after the immediately preceding dose.

In some embodiments, each dose is administered 2 weeks after theimmediately preceding dose.

In one embodiment, each dose comprises 1, 3 or 10 mg/kg of subject'sbody weight.

In another embodiment, said antibody or antigen-binding fragment thereofthat specifically binds PD-1 is an antibody as defined in any one of thepreceding embodiments.

In some embodiments, the present disclosure provides a method oftreating or inhibiting the growth of a tumor in a subject, the methodcomprising: selecting a subject with a brain cancer; and administeringto the subject in need thereof a therapeutically effective amount of anantibody or antigen-binding fragment thereof that specifically bindsPD-1.

In one embodiment, the subject has glioblastoma multiforme (GBM).

In another embodiment, the subject has newly diagnosed GBM.

In other embodiments, the subject is 65 years of age.

In some embodiments, the anti-PD-1 antibody or antigen-binding fragmentthereof is administered as one or more doses, wherein each dose isadministered 0.5 to 4 weeks after the immediately preceding dose.

In one embodiment, each dose is administered 2 weeks after theimmediately preceding dose.

In another embodiment, each dose comprises 1, 3 or 10 mg/kg of subject'sbody weight.

In other embodiments, the method further comprising administeringradiation therapy to the subject in need thereof.

In some embodiments, the radiation therapy is hypofractionated radiationtherapy.

In one embodiment, the subject is administered 20-50 Gy of radiation in2-20 fractions.

In another embodiment, the subject is administered radiation therapy 1week after the first dose of the anti-PD-1 antibody.

In other embodiments, the one or more doses of anti-PD-1 antibody arecomprised in one or more cycles of treatment, wherein each cyclecomprises 1-6 doses of the anti-PD-1 antibody.

In some embodiments, each cycle of treatment comprises 4 doses of theanti-PD-1 antibody, wherein each dose is administered 2 weeks after theimmediately preceding dose.

In one embodiment, each dose comprises 1, 3 or 10 mg/kg of subject'sbody weight.

In another embodiment, the first cycle of treatment further comprisesradiation therapy.

In other embodiments, the radiation therapy is hypofractionatedradiation therapy.

In some embodiments, the subject is administered 20-50 Gy of radiationin 2-20 fractions.

In one embodiment, the subject is administered 30 Gy in 5 dailyfractions.

In another embodiment, the radiation therapy is administered one weekafter the administration of the anti-PD-1 antibody.

In other embodiments, the method further comprises administering ananti-angiogenic agent to the subject if the subject developsintracranial edema following administration of the anti-PD-1 antibody.

In some embodiments, the anti-angiogenic agent is selected from thegroup consisting of a vascular endothelial growth factor (VEGF)inhibitor and an angiopoietin-2 (Ang-2) inhibitor.

In one embodiment, the anti-angiogenic agent is bevacizumab oraflibercept.

In other embodiments, said antibody or antigen-binding fragment thereofthat specifically binds PD-1 is an antibody as defined in any one of thepreceding embodiments.

EXAM PLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1 In Vivo Efficacy of Anti-PD-1 Antibody in Combination withRadiation Therapy Against MC38 Tumors

In this Example, the effect of PD-1 blockade in combination withradiation therapy was examined against established MC38 tumors in mice.

5×10⁵ MC38 colon carcinoma cells were implanted subcutaneously into theright flanks of female C57BL/6 mice (Jackson Laboratory). Treatment wasinitiated on day 9 post implantation when average tumor volumes reachedapproximately 100 mm³. The mice were randomly assigned to receive eitherisotype control (2A3, BioXcell) or PD-1 blocking antibody (RMP1-14,BioXCell) at 5 mg/kg, 2x a week, for a total of 5 intraperitonealinjections. One day post the start of antibody treatment, mice assignedto the radiotherapy groups received 12 Gy of irradiation to their rightflank tumors. Radiotherapy was delivered using the RS 2000 BiologicalResearch Irradiator (Rad Source) to anesthesized mice(ketamine/xylazine) shielded with partial body irradiation fixtures(Precision X-ray) and lead sheeting (Images Scientific Instruments).Tumor growth was evaluated 3x a week until days 70-80 when all mice wereeuthanized. FIG. 1 shows study design of the experiment which includesdosing of the anti-PD-1 antibody and radiation.

FIG. 2 and Table 1 show the average tumor volumes in mice administeredwith the anti-PD-1 antibody alone or in combination with radiation.

TABLE 1 Average tumor volumes in mice administered with anti-PD-1antibody alone or in combination with radiation Average tumor volume(mm³ ± SEM) Days post- Anti-PD-1 Isotype control + Anti-PD-1 antibody +implantation Isotype control antibody radiation radiation 4 15.39 ± 3.70 8.62 ± 3.02 13.28 ± 3.44 10.78 ± 3.01 7 41.11 ± 8.81 38.90 ± 7.09 49.86 ± 11.38 39.36 ± 6.32 8  68.64 ± 10.01  72.03 ± 12.13  74.03 ±14.83  73.70 ± 14.86 10 85.82 ± 4.10  94.98 ± 22.68 100.88 ± 11.46122.05 ± 15.05 14 725.87 ± 68.45  834.37 ± 206.70 320.10 ± 58.80 300.67± 60.74 16 1023.61 ± 191.41 1123.51 ± 310.04 276.17 ± 82.81 219.29 ±45.94 18 1573.64 ± 263.65 1710.30 ± 424.30  353.45 ± 121.47 250.17 ±74.70 21 2688.69 ± 502.39 2569.65 ± 633.35  494.53 ± 211.90  188.98 ±105.80 23  597.70 ± 267.02 141.37 ± 73.76 25  671.93 ± 347.76 134.87 ±75.67 28  879.64 ± 403.70 147.82 ± 70.88 30 1081.39 ± 426.80 133.13 ±88.88 32  177.73 ± 112.81 36  233.44 ± 152.91 39  258.23 ± 158.67 42 316.58 ± 160.91 44  332.73 ± 152.43 46  456.13 ± 209.45 49  564.05 ±262.32 51  925.92 ± 434.29 53  747.14 ± 350.90 56 1290.10 ± 584.62

PD-1 (RMP1-14) blockade synergized with local irradiation (XRT) andsignificantly induced tumor regression (4/6 mice) in MC38-tumor bearingmice, in comparison to XRT+isotype control treated mice (2/6 mice).Tumor growth was inhibited or delayed in mice treated with anti-PD-1antibody in combination with radiation. Mice treated with anti-PD-1antibody and radiation took more than 40 days to reach 500mm³ tumorvolume as compared to mice on monotherapy which took less than 20 daysto reach 500 mm³ tumor volume. Tumor regression was sustained for up to4 weeks for the combo (XRT+anti-PD-1 antibody) treated group (1 out ofthe 4 rejected tumors relapsed at this time point) versus 1.5 weeks forthe XRT+isotype treated group (1 out of the 2 rejected tumors relapsed).In this tumor model, PD-1 blockade as a monotherapy did not have aneffect on primary tumor growth.

TABLE 2 Percent survival of mice administered with anti-PD-1 antibodyalone or in combination with radiation days post Radiation + Radiation +implantation isotype Anti-PD-1 isotype anti-PD-1 4 100 100 100 100 7 100100 100 100 8 100 100 100 100 10 100 100 100 100 14 100 100 100 100 16100 100 100 100 18 100 100 100 100 21 100 100 100 100 23 100 83 100 10025 67 50 100 100 28 33 50 100 100 30 17 17 100 100 32 0 17 67 100 36 0 067 100 44 0 0 50 100 46 0 0 50 100 49 0 0 50 100 51 0 0 33 100 53 0 0 17100 56 0 0 17 100 58 0 0 17 83 60 0 0 17 50 63 0 0 17 50 65 0 0 17 50 810 0 17 50 85 0 0 17 50

The therapeutic efficacy of the combinatorial treatment (XRT+anti-PD-1antibody) was demonstrated by the statistically increased overallsurvival of this group (50% alive at 70 days post tumor implantation) incomparison to all other treatment groups: isotype control (0% alive atd70), anti-PD-1 antibody treatment (0% alive at d70), and XRT+isotypetreated mice (17% alive at d70) (FIG. 3 ; Table 2).

Example 2 In Vivo Efficacy of Anti-PD-1 Antibody and Radiation TherapyAgainst B16 Tumors

In this Example, the anti-tumor effect of anti-mouse PD-1 antibody incombination with radiation therapy was examined against established B16tumors in mice.

2×10⁵ B16F10.9 melanoma cells were implanted subcutaneously into theright flanks of female C57BLJ6 mice (Jackson Laboratory). Treatment wasinitiated when average tumor volumes reached approximately 150 mm³. Themice were randomly assigned to receive either isotype control (2A3,BioXcell) or PD-1 blocking antibody (RMP1-14, BioXCell) at 5 mg/kg, 2x aweek, for a total of 5 intraperitoneal injections. One day post thestart of antibody treatment, mice assigned to the radiotherapy groupsreceived 8 Gy of irradiation to their right flank tumors. Radiotherapywas delivered using the RS 2000 Biological Research Irradiator (RadSource) to anesthesized mice (ketamine/xylazine) shielded with partialbody irradiation fixtures (Precision X-ray) and lead sheeting (ImagesScientific Instruments). Tumor growth was evaluated 3x a week until days70-80 when all mice were euthanized. FIG. 4 shows study design of theexperiment which includes dosing of the anti-PD-1 antibody andradiation.

PD-1 (RMP1-14) blocking antibody treatment in combination with localirradiation (XRT) delayed B16 primary tumor growth in comparison to XRTor anti-PD-1 antibody monotherapy (FIG. 5 ; Table 3).

TABLE 3 Average tumor volumes in mice administered with anti-PD-1antibody alone or in combination with radiation Average tumor volume mm³± SEM Days post- Anti-PD-1 Isotype control + Anti-PD-1 antibody +implantation Isotype control antibody radiation radiation 6  5.75 ± 5.75 8.32 ± 8.32  13.79 ± 13.79  1.14 ± 0.85 8  55.98 ± 27.15  62.66 ± 15.80 57.18 ± 37.79  50.57 ± 38.33 12 157.34 ± 37.88 144.36 ± 37.81 237.84 ±71.27 177.91 ± 59.17 15 334.71 ± 61.71 193.32 ± 35.53  510.95 ± 171.15 372.53 ± 147.50 18  621.43 ± 136.09 363.80 ± 45.72  739.62 ± 244.10 440.33 ± 194.90 20  939.69 ± 158.50 561.64 ± 49.44  677.48 ± 175.75 486.35 ± 207.65 22 1329.77 ± 202.01  772.16 ± 118.26  759.15 ± 235.94 512.67 ± 220.30 25 2602.08 ± 434.08 1343.42 ± 120.65 1182.27 ± 336.32 789.80 ± 299.24 27 1533.03 ± 179.88 1321.13 ± 400.18  877.82 ± 296.5129 2104.46 ± 350.48  944.67 ± 333.16 33 1024.71 ± 321.20 36 1237.68 ±340.52

TABLE 4 Percent survival of mice administered with anti-PD-1 antibodyalone or in combination with radiation days post Radiation + Radiation +implantation isotype Anti-PD-1 isotype anti-PD-1 0 100 100 100 100 6 100100 100 100 8 100 100 100 100 12 100 100 100 100 15 100 100 100 100 18100 100 100 100 20 100 100 100 100 22 100 100 100 100 25 100 100 100 10027 40 100 100 100 29 0 100 80 100 33 0 80 80 100 36 0 20 60 100 39 0 060 83 41 0 0 60 83 43 0 0 20 67 46 0 0 20 67 48 0 0 20 50 50 0 0 0 50 530 0 0 33 55 0 0 0 33 57 0 0 0 33 60 0 0 0 17 62 0 0 0 17 64 0 0 0 17 660 0 0 0

Combination of XRT plus anti-PD-1 antibody treatment increased overallsurvival (50% alive at d50 post implantation) in comparison to XRT alone(0% alive by d50), anti-PD-1 antibody alone (0% alive by d40), andisotype alone (0% alive by d30) (FIG. 6 ; Table 4).

Example 3 In Vivo Efficacy of Anti-PD-1 Antibody in Combination withRadiation Therapy Against Metastatic Lung Tumors

In this Example, the effect of PD-1 blockade in combination withradiation therapy was examined against established and metastatic tumorsin mice.

1.5×10⁵ 4T1 mammary carcinoma cells were implanted subcutaneously intothe right flanks of female Balb/c mice (Jackson Laboratory). Treatmentwas initiated on day 12 post implantation when average tumor volumesreached approximately 100 mm³. The mice were randomly assigned toreceive either isotype control (2A3, BioXcell) or PD-1 blocking antibody(RMP1-14, BioXCell) at 5 mg/kg, 2x a week, for a total of 5intraperitoneal injections. One day post the start of antibodytreatment, mice assigned to the radiotherapy groups received 8 Gy ofirradiation to their right flank tumors. Radiotherapy was deliveredusing the RS 2000 Biological Research Irradiator (Rad Source) toanesthesized mice (ketamine/xylazine) shielded with partial bodyirradiation fixtures (Precision X-ray) and lead sheeting (ImagesScientific Instruments). Tumor growth was evaluated 3x a week until day28 when all mice were euthanized in order to evaluate lung metastaticburden using a clonogenic assay. Briefly, lung tissue was dissociatedwith DNAse/Liberase TL (Roche) and cultured in media supplemented with60 uM 6-thioguanine. After two weeks in culture, the plates werecounterstained with methylene blue and the number of colonies enumerated(one colony represents one metastatic 4T1 cell).

It is expected that treatment with anti-PD-1 antibody in combinationwith radiation promotes tumor regression as well as mediates suppressionof metastatic growth.

Example 4 In vivo efficacy of Anti-Human PD-1 Antibody in Combinationwith Radiation Therapy Promotes Abscopal Effect Against Distal Tumors

In this Example, the effect of PD-1 blockade in combination withradiation therapy was examined against primary and distal MC38 tumors inmice humanized for PD-1 using anti-human PD-1 antibodies.

The exemplary anti-PD-1 antibody used in this Example is REGN2810 (alsoknown as H4H7798N as disclosed in US20150203579), a fully humanmonoclonal anti-PD-1 antibody comprising a heavy chain comprising theamino acid sequence of SEQ ID NO: 9 and a light chain comprising theamino acid sequence of SEQ ID NO: 10; an HCVR/LCVR amino acid sequencepair comprising SEQ ID NOs: 1 /2; and heavy and light chain CDRsequences comprising SEQ ID NOs: 3-8.

Mice humanized for PD-1 were engineered using VelociGene® technology(Valenzuela et al 2003, Nat. Biotechnol. 21: 652-659; US PatentApplication Publication 2015/0366174).

5×10⁵ MC38 colon carcinoma cells were implanted subcutaneously intofemale humanized PD-1/C57BLJ6 mice on day 0 (primary tumor on rightflank) and day 3 (tumor on left flank; distal tumor). Treatment wasinitiated when the average primary tumor volumes reached approximately150 mm³. The mice were randomly assigned to receive either isotypecontrol or PD-1 blocking antibody (REGN2810) at 5 mg/kg, 2x a week, fora total of 8 intra-peritoneal injections. One day post the start ofantibody treatment, mice assigned to the radiotherapy groups received 8Gy of irradiation to their right flank tumors. Radiotherapy wasdelivered using the RS 2000 Biological Research Irradiator (Rad Source)to anesthetized mice (ketamine/xylazine) shielded with partial bodyirradiation fixtures (Precision X-ray) and lead sheeting (ImagesScientific Instruments). Primary and secondary tumor growth wasevaluated 3× a week until days 70-80 when all mice were euthanized. FIG.7 shows the study design of the experiment which includes dosing of theanti-PD-1 antibody and radiation.

Results

Primary Tumor: PD-1 blockade (REGN2810) treatment synergized with localirradiation (XRT) in rejecting primary MC38 tumors (4 out of 6 tumorfree mice) in comparison to XRT+isotype control treated mice (1/6 tumorfree mice). Tumor regression was sustained in the combo treated groupfor 8 weeks until end of experiment versus three weeks for theXRT+isotype treated group (the rejected tumor relapsed at this timepoint) (FIG. 8 ; Table 5).

TABLE 5 Average primary tumor volumes in mice administered with REGN2810alone or in combination with radiation Average tumor volume (mm³ ± SEM)Days post- Isotype control + REGN2810 + implantation Isotype controlREGN2810 radiation radiation 4  8.47 ± 5.22 13.86 ± 7.13  9.02 ± 3.07 3.75 ± 3.75 5 14.32 ± 4.76 22.08 ± 2.69 27.54 ± 4.90 10.00 ± 3.17 739.43 ± 5.36 35.47 ± 6.73 42.72 ± 8.00  32.80 ± 10.60 9  62.68 ± 12.03 84.73 ± 20.91  68.27 ± 11.65  47.26 ± 11.65 10 111.78 ± 24.45 108.15 ±27.17  96.18 ± 18.07  75.13 ± 11.56 11 147.89 ± 36.11 176.67 ± 43.99111.87 ± 10.12 110.27 ± 25.02 12 171.76 ± 41.23 154.97 ± 44.16 153.69 ±16.06 121.88 ± 29.86 14 304.95 ± 94.96 221.70 ± 65.96 147.22 ± 19.77144.71 ± 34.28 17  609.24 ± 227.64 296.69 ± 95.14 116.65 ± 27.03 135.26± 57.41 19  369.17 ± 128.37 114.85 ± 38.73 124.59 ± 55.50 21  442.13 ±158.80 127.77 ± 36.92 130.00 ± 78.30 24  641.92 ± 275.23 198.40 ± 67.81113.25 ± 74.51 26  896.32 ± 389.54 252.51 ± 98.39  116.90 ± 101.35 281200.99 ± 498.27  331.78 ± 125.55 120.05 ± 89.32 31  477.34 ± 181.97 168.62 ± 151.18 33  189.07 ± 154.91 35  164.70 ± 127.33 38  216.32 ±159.47 40  219.35 ± 156.39 42  292.37 ± 204.83

PD-1 blockade as a monotherapy mediated rejection in 2 out of 5 mice;however, 1 of the mice which rejected its primary tumor, succumbed tosecondary tumor growth, resulting in only 1 mouse surviving to the endof the experiment. The potent therapeutic efficacy of combinatorialtreatment (XRT+REGN2810) was demonstrated by statistically increasedoverall survival (-67% alive at 70 days post tumor implantation) incomparison to all other groups: isotype control or XRT alone (0% aliveat d70), and REGN2810 as a monotherapy (20% alive at d70) (FIG. 9 ;Table 6).

TABLE 6 Percent survival of mice administered with REGN2810 alone or incombination with radiation days post Radiation + Radiation +implantation isotype REGN2810 isotype REGN2810 0 100 100 100 100 4 100100 100 100 5 100 100 100 100 7 100 100 100 100 9 100 100 100 100 10 100100 100 100 11 100 100 100 100 12 100 100 100 100 14 100 100 100 100 17100 100 100 100 19 83 100 100 100 21 83 100 100 100 24 83 100 100 100 2650 100 100 100 28 33 100 100 100 31 33 67 100 100 33 33 33 83 100 35 3333 67 100 38 33 33 50 100 40 17 17 50 100 42 17 17 50 100 44 17 17 50100 49 0 17 17 83 54 17 0 83 56 17 0 83 59 17 0 83 61 17 0 67 63 17 0 67

Distal tumor: REGN2810 in combination with XRT significantly promoted anabscopal effect (rejection of a tumor implanted at a distal site) with 5out of 6 tumor free mice in comparison to XRT alone (2/6 distal tumorfree), REGN2810 alone (1/6 distal tumor free), and isotype controltreated mice (1/6 distal tumor free) (FIG. 10 ; Table 7).

TABLE 7 Average distal tumor volumes in mice administered with REGN2810alone or in combination with radiation Days Average tumor volume (mm³ ±SEM) post- Isotype implan- Isotype control + REGN2810 + tation controlREGN2810 radiation radiation 1 0 0 0 0 2 0 0 0 0 4 0 0 0 0 6 0 0 0 0 711.13 ± 11.13 0 0 0 8 20.01 ± 20.01 0 3.26 ± 3.26 0 9 25.43 ± 25.43 7.00± 7.00 9.75 ± 9.75 0 11 31.93 ± 29.32 42.24 ± 26.88 28.81 ± 17.01 12.13± 12.13 14 56.20 ± 34.46 59.40 ± 29.41 57.64 ± 29.91 20.93 ± 14.07 1658.64 ± 29.57 95.78 ± 52.87 14.03 ± 9.79  18 151.71 ± 76.86  115.16 ±59.43  22.87 ± 16.20 21 207.13 ± 128.83 227.22 ± 105.46 17.01 ± 17.01 23333.43 ± 220.57 335.13 ± 148.86 9.51 ± 9.51 25 506.55 ± 355.36 503.71 ±211.49 11.45 ± 11.45 28 968.92 ± 418.57 31.59 ± 31.59 30 57.40 ± 57.4032 83.94 ± 83.94 35 133.89 ± 133.89 37 224.65 ± 224.65

Example 5 In Vivo Efficacy of Anti-PD-1 Antibody in Combination withRadiation Therapy and a GITR Antagonist Against MC38 Tumors

In this Example, the effect of PD-1 blockade in combination withradiation therapy and a glucocorticoid-induced tumor necrosis factorreceptor (GITR) antagonist (an anti-GITR antibody) was examined againstlarge established MC38 tumors in mice.

5×10⁵ MC38 colon carcinoma cells were implanted subcutaneously into theright flanks of female C57BLJ6 mice (Jackson Laboratory). Treatment wasinitiated when average tumor volumes reached approximately 150-200 mm³(categorized as “large tumors”). The mice were randomly assigned toreceive either isotype control antibody (2A3 or LTF-2; BioXcell), ananti-PD-1 antibody (RMP1-14; BioXcell), an anti-GITR antibody (DTA-1;BioXcell), or the combination of both anti-PD-1 antibody and anti-GITRantibody at 5 mg/kg, 2x a week, for a total of 5 intraperitonealinjections. One day post the start of antibody treatment, mice assignedto the radiotherapy groups received 8 Gy of irradiation to their rightflank tumors. Radiotherapy was delivered using the RS 2000 BiologicalResearch Irradiator (Rad Source) to anesthesized mice(ketamine/xylazine) shielded with partial body irradiation fixtures(Precision X-ray) and lead sheeting (Images Scientific Instruments).Tumor growth was evaluated 3x a week until days 70-80 when all mice wereeuthanized. FIG. 11 shows study design of the experiment which includesdosing of the anti-PD-1 antibody, anti-GITR antibody, and radiation.

The anti-PD-1 antibody (RMP1-14) treatment synergized with localirradiation (XRT) and the anti-GITR antibody in rejecting large MC38tumors (4 out of 6 tumor free mice) in comparison to XRT+anti-GITRantibody (2/6 tumor free), XRT+anti-PD-1 antibody (2/6 rejected), or XRTalone (0/6 tumor free) treated mice. Monotherapy (with anti-PD-1antibody or anti-GITR antibody) or combinatorial treatment (anti-PD-1antibody+anti-GITR antibody) had minimal effect on tumor growth withanti-PD-1 antibody or anti-GITR antibody treatment mediating rejectionin 1/5 mice and the combination of the two antibodies mediatingrejection in 2/5 mice. Tumor regression was sustained for up to 6.5weeks after the start of treatment for the triple combo treated miceversus 2 weeks for the XRT+anti-GITR antibody treated mice (FIG. 12 ).

TABLE 8 Percent survival of mice administered anti-PD-1 antibody incombination with radiation and anti-GITR antibody Days post Radiation +implan- Anti-PD-1 + Radiation + Radiation + Radiation + anti-PD-1 +tation Isotype Anti-PD-1 Anti-GITR anti-GITR isotype anti-PD-1 anti-GITRanti-GITR 0 100 100 100 100 100 100 100 100 28 100 100 80 100 100 100100 100 32 80 80 80 80 100 100 100 100 35 60 60 40 60 100 80 83 100 3820 20 20 60 40 80 50 100 41 0 20 20 60 20 60 50 100 48 0 20 20 60 0 6050 83 56 0 20 20 40 0 40 33 67 66 0 20 20 40 0 40 17 67 77 0 20 20 40 040 17 67

Table 8 and FIG. 13 show the survival of mice administered withanti-PD-1 antibody in combination with radiation therapy and anti-GITRantibody. Further, administration of anti-PD-1 antibody+XRT led to tumorregression of very large tumors (-300 mm³).

Example 6 In Vivo Efficacy of Anti-PD-1 Antibody in Combination withRadiation Therapy and a GITR Antagonist Against B16 Tumors

In this Example, the effect of PD-1 blockade in combination withradiation therapy and a GITR antagonist (anti-GITR antibody) wasexamined against established B16 tumors in mice.

2.5×10⁵ B16F10.9 melanoma cells were implanted subcutaneously into theright flanks of female C57BL/6 mice (Jackson Laboratory). Treatment wasinitiated when average tumor volumes reached approximately 100 mm³. Themice were randomly assigned to receive either isotype controls (2A3,LTF-2; BioXcell), anti-PD-1 antibody (RMP1-14, BioXcell), anti-GITRantibody (DTA-1; BioXcell), or the combination of both the anti-PD-1antibody and anti-GITR antibody at 5 mg/kg, 2× a week, for a total of 5intraperitoneal injections. One day post the start of antibodytreatment, mice assigned to the radiotherapy groups received 8 Gy ofirradiation to their right flank tumors. Radiotherapy was deliveredusing the RS 2000 Biological Research Irradiator (Rad Source) toanesthesized mice (ketamine/xylazine) shielded with partial bodyirradiation fixtures (Precision X-ray) and lead sheeting (ImagesScientific Instruments). Tumor growth was evaluated 3x a week until days70-80 when all mice were euthanized.

It is expected that anti-PD-1 antibody in combination with the anti-GITRantibody and radiation therapy promotes more tumor regression and delayin tumor growth than monotherapy or ant-PD-1 antibody in combinationwith radiation therapy.

Example 7 Clinical Trial of Anti-PD-1 Antibody and Radiation Therapy inPatients with Advanced Solid Tumors

This study is an open-label, multicenter, dose escalation study withmultiple dose escalation and expansion arms to investigate the efficacy,safety, and tolerability of anti-PD-1 antibody alone and in combinationwith other anti-cancer therapies (including radiation therapy), in adultpatients with advanced solid tumors.

The exemplary anti-PD-1 antibody used in this study is REGN2810 (alsoknown as H4H7798N as disclosed in US20150203579), a fully humanmonoclonal anti-PD-1 antibody comprising a heavy chain comprising theamino acid sequence of SEQ ID NO: 9 and a light chain comprising theamino acid sequence of SEQ ID NO: 10; an HCVR/LCVR amino acid sequencepair comprising SEQ ID NOs: 1 /2; and heavy and light chain CDRsequences comprising SEQ ID NOs: 3-8.

Study Objectives

The primary objective of the study is to characterize the safety,tolerability, dose limiting toxicities (DLTs) of REGN2810 administeredintravenously (IV) as monotherapy, or in combination with targetedradiation (with the intent to have this serve as an immuno-stimulatory,rather than primarily tumor-ablative therapy), low-dose cyclophosphamide(a therapy shown to inhibit regulatory T-cell responses), granulocytemacrophage colony-stimulating factor, carboplatin, docetaxel, or acombination thereof in patients with advanced malignancies.

The secondary objectives of the study are: (1) to determine arecommended phase 2 dose (RP2D) of REGN2810 as monotherapy and incombination with other anti-cancer therapies (targeted radiation,low-dose cyclophosphamide, or both); (2) to describe preliminaryantitumor activity of REGN2810, alone and with each combination partner(s); (3) to characterize the PK of REGN2810 as monotherapy and incombination with other anti-cancer therapies (targeted radiation,low-dose cyclophosphamide, or both); and (4) to assess immunogenicity ofREGN2810.

Rationale for Study Design

The 3+3 model for the dose-escalation phase of this study is designed topermit evaluation of the safety of REGN2810, both as monotherapy atdifferent dose levels, and in combination with immune-enhancingtreatments: cyclophosphamide; limited, targeted radiation delivered in 1of 2 dosing regimens; or combined radiation and cyclophosphamide.

Once the tolerability of REGN2810 has been established alone and incombination with radiation and/or cyclophosphamide, multiple expansioncohorts using various combinations or monotherapy in select indications[NSCLC, BC, HNSCC, CSCC, tumors with MSI (colorectal, endometrial,prostate, or other tumor types), HCC, and other advanced solid tumors]are added in order to further confirm the safety and evaluate theaugmentation of antitumor activity. Granulocyte-macrophagecolony-stimulating factor (GM-CSF), carboplatin, and/or docetaxel areadded to some of these combinations.

Table 9 lists some of the cohorts using REGN2810 monotherapy and incombination with other treatment modalities.

TABLE 9 A list of some of the expansion cohorts for REGN2810 monotherapyand combination therapies Cohort Indication Treatment 1 Non-small-celllung cancer (NSCLC) Flat dose-200 mg REGN2810 2 NSCLC 3 mg/kg REGN2810 +radiotherapy (9 Gy × 3) 3 Head and neck squamous cell carcinoma 3 mg/kgREGN2810 + radiotherapy (9 Gy × 3) + (HNSCC) cyclophosphamide + GM-CSF 4Breast cancer (BC) 3 mg/kg REGN2810 + radiotherapy (9 Gy × 3) +cyclophosphamide 5 Advanced solid tumors-Previous treatment with 3 mg/kgREGN2810 + radiotherapy (9 Gy × 3) + an anti PD-1/PD-L1 antibodycyclophosphamide + GM-CSF 6 Advanced solid tumors (excluding NSCLC, 3mg/kg REGN2810 + radiotherapy (9 Gy × 3) + HNSCC, and BC)cyclophosphamide + GM-CSF 7 Metastatic (M1) cutaneous squamous cell 3mg/kg REGN2810 carcinoma (CSCC) 8 Locally and/or regionally advancedCSCC (M0) 3 mg/kg REGN2810 that is unresectable 9 Metastatic colorectalcancer with microsatellite 3 mg/kg REGN2810 instability (MSI) 10Metastatic endometrial cancer with MSI 3 mg/kg REGN2810 11 Castraterecurrent prostate cancer with MSI 3 mg/kg REGN2810 12 Any otheradvanced solid tumor with MSI 3 mg/kg REGN2810 13 Advanced or metastatichepatocellular cancer 3 mg/kg REGN2810 (HCC) 14 Advanced solid tumorrefractory to first line 3 mg/kg REGN2810 + carboplatin + docetaxelchemotherapy (low dose) 15 Advanced solid tumor refractory to first line3 mg/kg REGN2810 + docetaxel (low dose) chemotherapy 16 Metastaticcolorectal cancer with MSI, 3 mg/kg REGN2810 previously untreated 17Advanced NSCLC previously untreated 3 mg/kg REGN2810 + carboplatin +docetaxel (low dose) 18 Newly diagnosed glioblastoma multiforme REGN2810(1 or 3 mg/kg) + radiotherapy (GBM) (6 Gy × 5 days) 19 Recurrent GBMREGN2810 (1 or 3 mg/kg) + radiotherapy (6 Gy × 5 days) 20 HIV and solidtumors 3 mg/kg REGN2810 21 Advanced NSCLC, previously untreated 3 mg/kgREGN2810 + Carboplatin + Paclitaxel (Full Dose) 22 Advanced Non-SquamousNSCLC, previously 3 mg/kg REGN2810 + Cisplatin + Pemetrexed untreated 23Advanced Squamous NSCLC, previously 3 mg/kg REGN2810 + Cisplatin +Gemcitabine untreated 24 Cervical Cancer, recurrent or metastatic 3mg/kg REGN2810 25 Basal cell carcinoma, refractory to hedgehog 3 mg/kgREGN2810 pathway inhibition 26 Advanced Solid Tumor 3 mg/kg REGN2810

The initial planned treatment with REGN2810 is every 14 days for up to48 weeks, with 24 weeks of follow-up observation. Radiation isadministered a week after the first dose of REGN2810. Low-dosecyclophosphamide is administered to patients assigned tocyclophosphamide 1 day before each of the first 4 doses of REGN2810.

Study Duration

Patients receive up to 48 weeks of treatment, after which there is a 24week follow-up period. A patient receives treatment until the 48 weektreatment period is complete, or until disease progression, unacceptabletoxicity, withdrawal of consent, or meeting of another study withdrawalcriterion. After a minimum of 24 weeks of treatment, patients withconfirmed complete responses (CR) may elect to discontinue treatment andcontinue with all relevant study assessments (eg, efficacy assessments).After a minimum of 24 weeks of treatment, patients with tumor burdenassessments of stable disease (SD) or partial response (PR) that havebeen unchanged for 3 successive tumor evaluations may also elect todiscontinue treatment and continue with all relevant study assessments(e.g., efficacy assessments).

Study Population

The target population for this study comprises patients with advancedmalignancies who are not candidates for standard therapy, unwilling toundergo standard therapy, or for whom no available therapy is expectedto convey clinical benefit; and patients with malignancies that areincurable and have failed to respond to or showed tumor progressiondespite standard therapy.

Inclusion criteria: A patient must meet with the following criteria tobe eligible for inclusion in the study: (1) demonstrated progression ofa solid tumor with no alternative standard-of-care therapeutic optionavailable; (2) at least 1 lesion for response assessment. Patientsassigned to radiotherapy require at least one additional lesion that canbe safely irradiated while sparing the index lesions and for whichradiation at the limited, palliative doses contemplated would beconsidered medically appropriate; (3) patients must have relapsed after,or be refractory to first-line therapy (and up to 2 prior lines oftherapy) in the recurrent or metastatic disease setting and must havedisease for which palliative radiation therapy is indicated; (4)patients with metastatic cancer with microsatellite instability (MSI)refractory to up to 2 prior lines of therapy; (5) Eastern CooperativeOncology Group (ECOG) performance status 1; (6) more than 18 years old;(7) hepatic function: a. total bilirubin 1.5x upper limit of normal(ULN; if liver metastases 3× ULN), b. transaminases 3× ULN (or 5.0x ULN,if liver metastases), c. alkaline phosphatase (ALP) 2.5× ULN (or 5.0×ULN, if liver metastases); (8) renal function: serum creatinine 1.5×ULN; (9) neutrophil count (ANC) 1.5×10⁹/L, c. platelet count 75×10⁹/L;(10) ability to provide signed informed consent; and (11) ability andwillingness to comply with scheduled visits, treatment plans, laboratorytests, and other study-related procedures.

Study Treatments

REGN2810 is supplied as a liquid in sterile, single-use vials. Each vialcontains a volume sufficient to withdraw 10 mL of REGN2810 at aconcentration of 25 mg/mL. REGN2810 is administered in an outpatientsetting as a 30 minute IV infusion. Each patient's dose depends onindividual body weight. The dose of REGN2810 is adjusted each cycle forchanges in body weight of 10′)/0. REGN2810 is administered alone, or incombination with radiation and/or cyclophosphamide. Cyclophosphamide isadministered at 200mg/m2 or as a low dose (100 mg/m2).

Monotherapy

REGN2810 is administered in an outpatient setting by IV infusion over 30minutes every 14 days for 48 weeks (ie, Days 1, 15±3, 29±3, and 43±3 ofa 56 day cycle). Planned monotherapy regimens to be assigned mayinclude: (i) 1 mg/kg IV infusion over 30 minutes every 14 days for 48weeks; (ii) 3 mg/kg infusion over 30 minutes every 14 days for 48 weeks;(iii) 10 mg/kg infusion over 30 minutes every 14 days for 48 weeks; (iv)0.3 mg/kg infusion over 30 minutes every 14 days for 48 weeks (if MTD isdetermined to be below 1 mg/kg); and (v) 200 mg flat dose IV infusionover 30 minutes every 14 days for 48 weeks.

Combination Therapy

Concomitant radiation therapy, cyclophosphamide, GM-CSF, carboplatin,and docetaxel is supplied through a prescription and their usage, dose,dose modifications, reductions, or delays, as well as any potential AEsresulting from their use, is tracked along with that of REGN2810.

Co-administration of REGN2810 and radiation: REGN2810 is administered byIV infusion over 30 minutes every 14 days for 48 weeks in combinationwith radiation treatment from day 8 to day 12. Planned combinationREGN2810 and radiation therapy regimens may include:

1 mg/kg REGN2810 infusion over 30 minutes every 14 days for 48 weeksplus 30 Gy radiotherapy (6 Gy x 5 times/week; given 1 week after thefirst dose of REGN2810, preferably on consecutive days) 1 mg/kg REGN2810infusion over 30 minutes every 14 days for 48 weeks plus 27 Gyradiotherapy (9 Gy x 3 times/week; given 1 week after the first dose ofREGN2810, preferably not on consecutive days) 3 mg/kg REGN2810 infusionover 30 minutes every 14 days for 48 weeks plus 30 Gy radiotherapy (6 Gyx 5 times/week; given 1 week after the first dose of REGN2810,preferably on consecutive days) 3 mg/kg REGN2810 infusion over 30minutes every 14 days for 48 weeks plus 27 Gy radiotherapy (9 Gy x 3times/week; given 1 week after the first dose of REGN2810, preferablynot on consecutive days)

Patients will receive either 30 Gy given as 5 fractions of 6 Gyadministered daily starting 1 week after the first dose of REGN2810, or27 Gy given as 3 fractions of 9 Gy administered every other day starting1 week after the first dose of REGN2810. The lesion selected forradiation should be a lesion that can be safely irradiated with focalirradiation while sparing the index lesion(s), and for which radiationat the limited, palliative doses contemplated would be consideredmedically appropriate.

Co-administration of REGN2810 and cyclophosphamide: REGN2810 isadministered by IV infusion over 30 minutes every 14 days (2 weeks) for48 weeks in combination with low dose cyclophosphamide 100 mg/m2 IVinfusion every 14 days for 4 doses. Each of the 4 cyclophosphamide dosesare administered 1 day before each of the first 4 REGN2810 doses (days—1, 14, 28, and 42 of the first 56 day cycle).

The planned combination REGN2810 and cyclophosphamide regimen is:

Cyclophosphamide 100 mg/m2 or 200 mg/m2 IV every 14 days (days —1, 14,28, and 42 of the first 56 day cycle) for a total of 4 doses; plus 3mg/kg REGN2810 infusion over 30 minutes every 14 days for 48 weeks(provided monotherapy dose of 3 mg/kg <MTD; if 3 mg/kg >MTD, dose willbe 1 mg/kg.

Co-administration of REGN2810, radiation and cyclophosphamide: Theplanned combination REGN2810, radiation, and cyclophosphamide regimenincludes:

Cyclophosphamide 100 mg/m2 (low dose) IV every 14 days (days —1, 14, 28,and 42 of the first 56 day cycle) for a total of 4 doses; plus 27 Gyradiotherapy (9 Gy x 3 times/week; given 7 or 8 days after the firstdose of

REGN2810, preferably not on consecutive days) OR 30 Gy radiotherapy (6Gy x 5 times/week; given 7 or 8 days after the first dose of REGN2810,preferably on consecutive days); plus 3 mg/kg REGN2810 infusion over 30minutes every 14 days for 48 weeks (provided monotherapy dose of 3 mg/kg<MTD; if 3 mg/kg >MTD, dose will be 1 mg/kg)

Co-administration of REGN2810, radiation and GM-CSF: The plannedcombination REGN2810, radiation, and GM-CSF regimen includes:

GM-CSF 250 mcg SC daily for 7 days, for four 7-day intervals (days 1through 7, 15 through 21, 29 through 35, and 43 through 49 of the first56-day cycle); plus 27 Gy radiotherapy (9 Gy x 3 times/week; given1-week after the first dose of REGN2810, preferably not on consecutivedays); plus 3 mg/kg REGN2810 infusion over 30 minutes every 14 days for48 weeks (provided monotherapy dose of 3 mg/kg <MTD; if 3 mg/kg >MTD,dose will be 1 mg/kg)

Co-administration of REGN2810, radiation, GM-CSF and cyclophosphamide:The planned combination REGN2810, radiation, GM-CSF, andcyclophosphamide regimen includes:

GM-CSF 250 mcg SC daily for 7 days, for four 7-day intervals (days 1through 7, 15 through 21, 29 through 35, and 43 through 49 of the first56-day cycle); plus 27 Gy radiotherapy (9 Gy x 3 times/week; given 1week after the first dose of REGN2810, preferably not on consecutivedays); plus Cyclophosphamide 100 mg/m2 or 200 mg/m2 IV every 14 days(days —1, 14, 28, and 42 of the first 56 day cycle) for a total of 4doses; plus 3 mg/kg REGN2810 infusion over 30 minutes every 14 days for48 weeks (provided monotherapy dose of 3 mg/kg <MTD; if 3 mg/kg >MTD,dose will be 1 mg/kg)

Co-administration of REGN2810 and docetaxel with or without carboplatin:The suggested sequence of drug administration is docetaxel followed bycarboplatin (if enrolled in a carboplatin-containing cohort), followedby REGN2810:

Docetaxel 30 mg/m2 IV over approximately 1 hour on days 1, 8, 29, and 36of the first 56-day cycle. Dexamethasone 8 mg IV will be administeredprior to the first dose of docetaxel. For subsequent docetaxeltreatments, the dose of dexamethasone premedication may be 8 mg or 4 mg,per investigator discretion Carboplatin AUC 2 IV over approximately 30minutes on days 1, 8, 29, and 36 of the first 56-day cycle. Carboplatindosing should use the Calvert formula on the carboplatin label.Creatinine clearance should be calculated using the Cockcroft-Gaultequation. 3 mg/kg REGN2810 infusion over approximately 30 minutes every14 days for 48 weeks

Procedures and Assessments

Screening procedures to be performed include serum beta-HCG, brain MRI,and chest X-rays.

Safety procedures include medical history, physical examination, vitalsigns, electrocardiogram (ECG), coagulation, immune safety assays (forpatients treated with REGN2810), assessment of B symptoms and evaluationof performance status, clinical laboratory tests, AEs, and concomitantmedications.

Efficacy procedures to be performed for tumor assessments include CT orMRI scans, 18F-fluorodeoxyglucose-positron emission tomography (FDG-PET)scans, and/or tumor biopsies. A CT or MRI for tumor assessment isperformed at the screening visit (within 28 days prior to infusion) andduring every cycle (approximately every 8 weeks) on day 56±3, and whendisease progression is suspected. Additionally, for patients who havenot progressed on study, tumor assessments are performed for follow-upvisits 3, 5, and 7. Once the choice has been made to use CT scan or MRI,subsequent assessments are made using the same modality. Tumor responseassessments are performed according to Response Evaluation Criteria inSolid Tumors RECIST version 1.1 (Eisenhauer et al 2009, Eur. J. Cancer45: 228-247). Measurable lesions selected as target lesions for RECISTmeasurements are also included as index lesions for immune-relatedresponse criteria (irRC; Nishino et al 2013, Clin. Cancer Res. 19:3936-3943). RECIST response is prioritized as statistical assessment ofresponse rate. For an individual patient, irRC can inform the decisionregarding whether to continue treatment at the discretion of theinvestigator due to the possibility of unconventional responses.

Blood samples for PK and anti-drug antibody (ADA) assessment arecollected.

Study Variables

The primary variables in the study are DLT incidence and the incidenceand severity of TEAEs and abnormal laboratory findings through 48 weeksof treatment.

The secondary variables are:

Antitumor activities assessed using the appropriate criteria for theindication (described elsewhere herein):

-   -   Response Evaluation Criteria in Solid Tumors (RECIST; Eisenhauer        et al 2009, Eur. J. Cancer 45: 228-247) criteria measured by CT        or MRI    -   Other assessment criteria also are used for specific tumors in        which RECIST measurements are not the standard.    -   Immune-Related Response Criteria (irRC; Nishino et al 2013,        Clin. Cancer Res. 19: 3936-3943) applied to RECIST measurements.        In all cases, RECIST (or other tumor-specific criteria) is the        governing tool to determine PD, SD, CR, or PR. The irRC is        collected for clinical decisions and information purposes.

Incidence of development of anti-REGN2810 antibodies

Antitumor activity measured by PFS and overall survival

For the purposes of this study, patients are re-evaluated for responseevery 8 weeks. Confirmatory scans are also obtained 4 weeks followinginitial documentation of objective response or progressive disease.Response and progression is evaluated in this study using theinternational criteria proposed by the revised Response EvaluationCriteria in Solid Tumors (RECIST) guideline (version 1.1; Eisenhauer etal 2009, Eur. J. Cancer 45: 228-247). Changes in the largest diameter(unidimensional measurement) of the tumor lesions and the shortestdiameter in the case of malignant lymph nodes are used in the RECISTcriteria.

Selection of Lesions

Measurable disease: Measurable lesions are defined as those that can beaccurately measured in at least one dimension (longest diameter to berecorded) as 20 mm (2 cm) by chest x-ray or as 0 mm (1 cm) with CT scan,MRI, or calipers by clinical exam. All tumor measurements must berecorded in millimeters (or decimal fractions of centimeters). Note: Seebelow for evaluation of radiated target lesions.

Malignant lymph nodes: To be considered pathologically enlarged andmeasurable, a lymph node must be mm (1.5 cm) in short axis when assessedby CT scan (CT scan slice thickness recommended to be no greater than 5mm [0.5 cm]). At baseline and in follow-up, only the short axis will bemeasured and followed.

Non-measurable disease: All other lesions (or sites of disease),including small lesions (longest diameter <10 mm [<1 cm] or pathologicallymph nodes with to <15 mm [1 to <1.5 cm] short axis), are considerednon-measurable disease. Bone lesions, leptomeningeal disease, ascites,pleural/pericardial effusions, lymphangitis cutis/pulmonitis,inflammatory breast disease, and abdominal masses (not followed by CT orMRI), are considered as non-measurable. Note: Cystic lesions that meetthe criteria for radiographically defined simple cysts should not beconsidered as malignant lesions (neither measurable nor non-measurable)since they are, by definition, simple cysts. ‘Cystic lesions’ thought torepresent cystic metastases can be considered as measurable lesions, ifthey meet the definition of measurability described above. However, ifnon-cystic lesions are present in the same patient, these are preferredfor selection as target lesions.

Target lesions: All measurable lesions up to a maximum of 2 lesions perorgan and 5 lesions in total, representative of all involved organs,should be identified as target lesions and recorded and measured atbaseline. Target lesions are selected on the basis of their size(lesions with the longest diameter), are representative of all involvedorgans, but in addition include those that lend themselves toreproducible repeated measurements. It may be the case that, onoccasion, the largest lesion does not lend itself to reproduciblemeasurement in which circumstance the next largest lesion which can bemeasured reproducibly is selected. A sum of the diameters (longest fornon-nodal lesions, short axis for nodal lesions) for all target lesionsis calculated and reported as the baseline sum diameters. If lymph nodesare to be included in the sum, then only the short axis is added intothe sum. The baseline sum diameters are used as reference to furthercharacterize any objective tumor regression in the measurable dimensionof the disease.

Non-target lesions: All other lesions (or sites of disease) includingany measurable lesions over and above the 5 target lesions areidentified as non-target lesions and are recorded at baseline.Measurements of these lesions are not required, but the presence,absence, or in rare cases unequivocal progression of each is notedthroughout follow-up.

Methods for Evaluation of Measurable Disease

All measurements are taken and recorded in metric notation using a ruleror calipers. All baseline evaluations are performed as closely aspossible to the beginning of treatment and never more than 4 weeksbefore the beginning of the treatment. The same method of assessment andthe same technique should be used to characterize each identified andreported lesion at baseline and during follow-up. Imaging-basedevaluation is preferred to evaluation by clinical examination unless thelesion(s) being followed cannot be imaged but are assessable by clinicalexam.

Clinical lesions: Clinical lesions are only considered measurable whenthey are superficial (eg, skin nodules and palpable lymph nodes) and mm(1 cm) diameter as assessed using calipers (e.g., skin nodules). In thecase of skin lesions, documentation by color photography, including aruler to estimate the size of the lesion, is recommended.

Chest x-ray: Lesions on chest x-ray are acceptable as measurable lesionswhen they are clearly defined and surrounded by aerated lung. However,CT is preferable.

Conventional CT and MRI: This guideline has defined measurability oflesions on CT scan based on the assumption that CT slice thickness is 5mm (0.5 cm) or less. If CT scans have slice thickness greater than 5 mm(0.5 cm), the minimum size for a measurable lesion should be twice theslice thickness. MRI is also acceptable in certain situations.

PET-CT: If the CT performed as part of a PET-CT is of identicaldiagnostic quality to a diagnostic CT (with IV and oral contrast), thenthe CT portion of the PET-CT can be used for RECIST measurements and canbe used interchangeably with conventional CT in accurately measuringcancer lesions over time.

Ultrasound: Ultrasound is not useful in assessment of lesion size andshould not be used as a method of measurement. If new lesions areidentified by ultrasound in the course of the study, confirmation by CTor MRI is advised. If there is concern about radiation exposure at CT,MRI may be used instead of CT in selected instances.

Endoscopy, Laparoscopy: The utilization of these techniques forobjective tumor evaluation is not advised. However, such techniques maybe useful to confirm complete pathological response when biopsies areobtained or to determine relapse in trials where recurrence followingcomplete response (CR) or surgical resection is an endpoint.

Tumor markers: Tumor markers alone cannot be used to assess response. Ifmarkers are initially above the upper normal limit, they must normalizefor a patient to be considered in complete clinical response.

Cytology, Histology: These techniques can be used to differentiatebetween partial responses (PR) and complete responses (CR) in rare cases(eg, residual lesions in tumor types, such as germ cell tumors, whereknown residual benign tumors can remain). The cytological confirmationof the neoplastic origin of any effusion that appears or worsens duringtreatment when the measurable tumor has met criteria for response orstable disease is mandatory to differentiate between response or stabledisease (an effusion may be a side effect of the treatment) andprogressive disease.

FDG-PET: While FDG-PET response assessments need additional study, it issometimes reasonable to incorporate the use of FDG-PET scanning tocomplement CT scanning in assessment of progression (particularlypossible ‘new’ disease). New lesions on the basis of FDG-PET imaging canbe identified according to the following algorithm: a. Negative FDG-PETat baseline, with a positive FDG-PET at follow-up is a sign of PD basedon a new lesion. b. No FDG-PET at baseline and a positive FDG-PET atfollow-up: If the positive FDG-PET at follow-up corresponds to a newsite of disease confirmed by CT, this is PD. If the positive FDG-PET atfollow-up is not confirmed as a new site of disease on CT, additionalfollow-up CT scans are needed to determine if there is truly progressionoccurring at that site (if so, the date of PD will be the date of theinitial abnormal FDG-PET scan). If the positive FDG-PET at follow-upcorresponds to a pre-existing site of disease on CT that is notprogressing on the basis of the anatomic images, this is not PD. c.FDG-PET may be used to upgrade a response to a CR in a manner similar toa biopsy in cases where a residual radiographic abnormality is thoughtto represent fibrosis or scarring. The use of FDG-PET in thiscircumstance should be prospectively described in the protocol andsupported by disease-specific medical literature for the indication.However, it must be acknowledged that both approaches may lead to falsepositive CR due to limitations of FDG-PET and biopsyresolution/sensitivity. Note: A ‘positive’ FDG-PET scan lesion means onewhich is FDG avid with an uptake greater than twice that of thesurrounding tissue on the attenuation corrected image.

Response Criteria for Evaluation of Tarciet Lesions

Complete Response (CR): Disappearance of all target lesions. Anypathological lymph nodes (whether target or non-target) must havereduction in short axis to <10 mm (<1 cm).

Partial Response (PR): At least a 30% decrease in the sum of thediameters of target lesions, taking as reference the baseline sumdiameters.

Progressive Disease (PD): At least a 20% increase in the sum of thediameters of target lesions, taking as reference the smallest sum onstudy (this includes the baseline sum if that is the smallest on study).In addition to the relative increase of 20%, the sum must alsodemonstrate an absolute increase of at least 5 mm (0.5 cm). (Note: theappearance of one or more new lesions is also considered progressions).

Stable Disease (SD): Neither sufficient shrinkage to qualify for PR norsufficient increase to qualify for PD, taking as reference the smallestsum diameters while on study.

Response Criteria for Evaluation of Non-Tarciet Lesions

Complete Response (CR): Disappearance of all non-target lesions andnormalization of tumor marker level. All lymph nodes must benon-pathological in size (<10 mm [<1 cm] short axis). Note: If tumormarkers are initially above the upper normal limit, they must normalizefor a patient to be considered in complete clinical response.

Non-CR/Non-PD: Persistence of one or more non-target lesion(s) and/ormaintenance of tumor marker level above the normal limits.

Progressive Disease (PD): Appearance of one or more new lesions and/orunequivocal progression of existing non-target lesions. Unequivocalprogression should not normally trump target lesion status. It must berepresentative of overall disease status change, not a single lesionincrease.

Immune-Related Response Criteria

Immune-related response criteria differ from RECIST (Version 1.1) inthat the sum of the longest diameters of all target lesions and newlesions if any are used to determine response. The presence of newlesions per se does not determine progression; the total tumor burden isconsidered.

Evaluation of Target Lesions

Complete Response (CR): Disappearance of all target lesions. Anypathological lymph nodes (whether target or non-target) must havereduction in short axis to <10 mm (<1 cm).

Partial Response (PR): At least a 30% decrease in the sum of thediameters of target lesions, including new lesions, taking as referencethe baseline sum diameters.

Progressive Disease (PD): At least a 20% increase in the sum of thediameters of target lesions, including new lesions, taking as referencethe smallest sum on study (this includes the baseline sum if that is thesmallest on study). In addition to the relative increase of 20%, the summust also demonstrate an absolute increase of at least 5 mm (0.5 cm).

Stable Disease (SD): Neither sufficient shrinkage to qualify for PR norsufficient increase to qualify for PD, taking as reference the smallestsum diameters while on study and including the measurements of newlesions.

Evaluation of Non-Target Lesions

Complete Response (CR): Disappearance of all non-target lesions andnormalization of tumor marker level. All lymph nodes must benon-pathological in size (<10 mm [<1 cm] short axis). Note: If tumormarkers are initially above the upper normal limit, they must normalizefor a patient to be considered in complete clinical response.

Non-CR/Non-PD: Persistence of one or more non-target lesion(s) and/ormaintenance of tumor marker level above the normal limits.

Progressive Disease (PD): Unequivocal progression of existing non-targetlesions. Unequivocal progression should not normally trump target lesionstatus. It must be representative of overall disease status change, nota single lesion increase. Although a clear progression of “non-target”lesions only is exceptional, the opinion of the treating physicianshould prevail in such circumstances, and the progression status shouldbe confirmed at a later time.

Evaluation of Overall Response Criteria

The best overall response is the best response recorded from the startof the treatment until disease progression/recurrence (taking asreference for progressive disease the smallest measurements recordedsince the treatment started). The patient's best response assignmentwill depend on the achievement of both measurement and confirmationcriteria. Revised Response Evaluation Criteria in Solid Tumors (RECIST)Version 1.1 (Eisenhauer et al 2009, Eur. J. Cancer 45: 228-247) andimmune-related response criteria (irRC; Nishino et al 2013, Clin. CancerRes. 19: 3936-3943) are summarized in Tables 10 and 11 below.

TABLE 10 Response according to Revised RECIST (Version 1.1) Target NewOverall Best Overall Response when Lesions Non-target Lesions LesionsResponse Confirmation is Required CR CR No CR ≥4 weeks confirmation CRNon-CR/Non-PD No PR ≥4 weeks confirmation CR Not evaluated No PR ≥4weeks confirmation PR Non-CR/Non-PD/not evaluated No PR ≥4 weeksconfirmation SD Non-CR/Non-PD/not evaluated No SD Documented at leastonce ≥4 weeks from baseline PD Any Yes or No PD No prior SD, PR or CRAny PD Yes or No PD No prior SD, PR or CR Any Any Yes PD No prior SD, PRor CR CR: complete response; PD: progressive disease; PR: partialresponse; SD: stable disease

TABLE 11 Immune-related Response Criteria Evaluation Target New OverallBest Overall Response when Lesions Non-target Lesions Lesions ResponseConfirmation is Required CR CR No CR ≥4 weeks confirmation CRNon-CR/Non-PD No PR ≥4 weeks confirmation CR Not evaluated No PR ≥4weeks confirmation PR Non-CR/Non-PD/not evaluated Yes or No PR ≥4 weeksconfirmation SD Non-CR/Non-PD/not evaluated Yes or No SD Documented atleast once ≥4 weeks from baseline PD Any Yes or No PD No prior SD, PR orCR Any PD Yes or No PD No prior SD, PR or CR CR: complete response; PD:progressive disease; PR: partial response; SD: stable disease

Evaluation of Radiated Tarciet Lesions

Radiated target lesions are evaluated with a modified version of theinternational criteria proposed by the Response Evaluation Criteria inSolid Tumors (RECIST) Committee, version 1.1. Additional definitionsbeyond the RECIST 1.1 guidelines specific to this protocol areincorporated to define local control.

The response criteria for radiated lesions are as follows:

Local enlargement (LE): At least a 20% increase in the LD of targetlesion, taking as reference the smallest LD recorded since the treatmentstarted. Ideally, this determination will be made based on CT imageevaluation.

Local failure (LF): Refers to the primary treated tumor after protocoltherapy and corresponds to meeting both of the following two criteria:(1) Increase in tumor dimension of 20% as defined above for localenlargement (LE); (2) The measurable tumor with criteria meeting LEshould be avid on Positron Emission Tomography (PET) imaging with uptakeof a similar intensity as the pretreatment staging PET, OR themeasurable tumor should be biopsied confirming viable carcinoma.

Local control (LC): The absence of local failure.

The longest diameter (LD) for the radiated target lesion calculated fromthe treatment-planning CT scan, using appropriate tissue-specificwindowing, is reported as the baseline LD. The baseline LD is used asthe reference by which to characterize the objective tumor. For follow-up assessment, diagnostic CT scans performed using a 5 mm contiguousreconstruction algorithm using pulmonary windowing taken as part ofscheduled protocol follow-up are preferred as the method of evaluationfor response. When CT scans are not available, MRI or x-raydetermination is allowed, as long as the target lesion is clearlyvisible.

Results

REGN2810 alone and in combination is safe and well-tolerated bypatients. Administration of REGN2810 alone or in combination with othertreatment modalities inhibits tumor growth and/or promotes tumorregression in patients with advanced solid tumors. Overall response rateis better for combination therapy with radiation as compared tomonotherapy.

60 patients with advanced solid malignancies (47% with four or moreprior therapies) have been treated to-date. The advanced solidmalignancies include colorectal cancer, head and neck cancer, breastcancer, soft tissue sarcoma, adrenal cancer, anal cancer, cancer of theappendix, bladder cancer, cervical cancer, endometrial cancer,esophageal cancer, liver cancer, non-small cell lung adenocarcinoma,ovarian cancer, pancreatic cancer, prostate cancer, renal sarcomatoid,salivary gland cancer, non-melanoma skin cancer, Merkel cell carcinoma,squamous cell carcinoma, basal cell carcinoma, small intestine cancer,thyroid cancer and uterine cancer.

Forty-two patients (70%) experienced one or more treatment-relatedadverse events (AEs). The most common treatment-related AEs were fatigue(28.3%), arthralgia (11.7%) and nausea (11.7%). Of the 60 patientsevaluated for tumor responses, there were 11 (18.3%) objective responses(PR/CR), while 31 patients (51.7%) showed disease control (CR/PR/SD). Inthe 36 patients who received combination therapy including radiationtherapy, objective response was seen in 6 patients (16.7%) and diseasecontrol in 19 patients (52.8%). In the 24 patients who did not receiveradiation therapy, objective response was seen in five patients (20.8%)and disease control was seen in 12 patients (50%). Table 12 shows asummary of responders.

TABLE 12 Summary of responders No. Prior Lines Subject of Best Best % IDDose Cohort Cancer Type Therapy Response Reduction 41 R2810: 1 mg/kgCholangiocarcinoma 5 PR −41.2 50 R2810: 1 mg/kg Cutaneous squamous cellcarcinoma 2 CR −100.0 43 R2810: 10 mg/kg Soft tissue sarcoma 5 PR −49.137 R2810: 10 mg/kg Basal cell carcinoma 1 PR −36.7 36 R2810: 3 mg/kg +CPA: 200 mg/m2 Soft tissue sarcoma 5 PR −33.3 47 R2810: 1 mg/kg + XRT: 6Gy × 5 Cervix squamous cell carcinoma 4 PR −66.7 46 R2810: 1 mg/kg +XRT: 9 Gy × 3 Anal squamous cell carcinoma 3 PR −57.1 49 R2810: 1mg/kg + XRT: 9 Gy × 3 Cervix squamous cell carcinoma 3 CR −100.0 48R2810: 3 mg/kg + XRT: 6 Gy × 5 Merkel Cell Carcinoma 1 PR −72.5 42R2810: 3 mg/kg + XRT: 6 Gy × 5 Small intestine adenocarcinoma 2 PR −46.744 R2810: 3 mg/kg + XRT: 9 Gy × 3 Ovarian serous carcinoma 6 PR −52.4

Among the responders, the median time to response for monotherapy was113 days (range 52-226) and for patients with radiation therapy was 59days (range 56-113).

Example 8 Case Reports of PD-1 Blockade with Monoclonal AntibodyREGN2810 Achieving Durable Objective Responses In Metastatic,Non-Melanoma Skin Cancers: Basal Cell Carcinoma and Cutaneous SquamousCell Carcinoma Introduction

Basal cell carcinoma (BCC) and cutaneous squamous cell carcinoma (CSCC)share exposure to UV light as the dominant risk factor, and these tumorsare therefore hypermutated (Chalmers et al 2016, AACR Ann. Meeting, Abs3576). In other malignancies, high mutation burden has been associatedwith clinical benefit from therapy with antibodies directed against thePD-1 immune checkpoint [Le et al 2015, New Engl. J. Med. May 30 (Epubahead of print)]. Highly mutated tumors are more likely to expressimmunogenic tumor neoantigens that attract effector T cells that can beunleashed by blockade of the PD-1 immune checkpoint (Mandal and Chan2016, Cancer Discov. 6: 1-12). This Example describes a patient withmetastatic BCC and a patient with metastatic CSCC who were treated withREGN2810, a fully human anti-PD-1 monoclonal antibody in an ongoingphase 1 trial (NCT02383212; described in Example 7 herein).

Case Report 1

The patient was a 66 year-old woman who was diagnosed with a stage 1 BCCarising on the left aspect of the chin, which was resected with Mohssurgery. A localized recurrence in the same location was identified 2years later, and a wide local excision revealed invasion into the leftmandible and involvement of one out of 18 lymph nodes. The patientreceived adjuvant radiation and remained in remission for 4 years, whenenlarging lung nodules observed on surveillance chest imaging werebiopsied and confirmed the presence of metastatic BCC. The patientsubsequently received the Hedgehog pathway inhibitor (HHI) vismodegibfor 5 months. She initially responded but discontinued because ofprogressive disease.

Six months after the vismodegib therapy and upon continued slowprogression, the patient enrolled on the phase 1 study of REGN2810 to acohort receiving 10 mg/kg IV every 2 weeks, and received her first dose.Two lung metastases were followed as target lesions. Responseassessments at the end of 8 weeks (3% increase) and 16 weeks (10%decrease) demonstrated stable disease by RECIST criteria. The responseassessment at the end of 24 weeks demonstrated a reduction in tumormeasurements of 37% (FIG. 14A), and this was confirmed at 32 weeks. Thepatient has tolerated treatment well, and continues REGN2810, ontreatment for 10+months.

Case Report 2

The patient was a 52 year-old man who was diagnosed with cutaneoussquamous cell carcinoma of the left cheek. He underwent Mohs surgerywith clear margins. He experienced multiple recurrences, and underwentat least 9 additional Mohs surgeries. He underwent wide local excisionover left mandible 4 years later, and left parotidectomy subsequently in20 months. Also, adjuvant radiotherapy was administered to left cheek,left mandible, left neck (with concurrent cetuximab), and bilateral neck(with concurrent carboplatin). Other systemic therapies werecapecitabine, and cisplatin+docetaxel. Ten years after the initialdiagnosis, he underwent excision with clear margins for a 2.2cm in-scarrecurrence of the left neck. Subsequently, invasive LSCC at C4-05vertebral bodies necessitated emergent decompression of cervical spinalcord with C4-05 anterior corpectomy and C4-C6 posterior laminectomy. Healso developed lower extremity muscle weakness thought to be due toperineural involvement and required the use of a walker for ambulation.

He was enrolled on the phase 1 study in the first cohort, receiving 1mg/kg REGN2810 every two weeks. Within weeks of beginning treatment, hislower extremity strength gradually returned and he no longer requiresthe use of the walker. Response at Week 16 is shown in FIG. 14B.Complete radiologic response of the left neck lesion was achieved atWeek 40. The patient completed the planned 48 weeks of protocoltreatment with REGN2810. He continues in close active follow up with hismedical oncologist without clinical or radiographic evidence of diseaserecurrence.

Discussion

This Example discloses the first confirmed partial response in a patientwith metastatic BCC treated with a PD-1 inhibitor (REGN2810), as well asan ongoing durable complete response in a patient with metastatic LSCC.The deep and sustained responses of these heavily pretreated patients toanti-PD-1 monotherapy in this phase 1 study are consistent with thehypothesis that high mutation burden in BCC and CSCC would elicitantitumor cellular immunity that could be unleashed by blockade of thePD-1/PD-L1 checkpoint pathway.

This Example supports a general principle that UV-associated skincancers beyond melanoma are sensitive to PD-1 blockade. A reductionistmodel would predict that UV-associated tumors with higher load ofnon-synonymous mutations will be more responsive to PD-1 blockade thanthose with lower mutation load.

Example 9 Safety and Efficacy of Anti-PD-1 Antibody in Patients withUnresectable Locally Advanced or Metastatic Cutaneous Squamous CellCarcinoma (CSCC) Background

There is no established standard of care for unresectable locallyadvanced or metastatic CSCC. Due to UV-induced DNA damage, most CSCCsare hyper-mutated. Therefore, these tumors may be responsive to PD-1checkpoint blockade. This Example describes patients with locallyadvanced or metastatic CSCC who were treated with REGN2810, a fullyhuman anti-PD-1 monoclonal antibody in an ongoing phase 1 trial(NCT02383212; described in Example 7 herein).

Methods

Expansion cohorts (ECs) in the phase 1 study of REGN2810 enrolledpatients with distantly metastatic CSCC (EC 7) and locally advanced CSCC(EC8) (Table 9). All patients received 3 mg/kg REGN2810 by vein every 2weeks for up to 48 weeks. Research biopsies were performed at baselineand Day 29 (and at progression, if possible). To determine overallresponse rate, tumor measurements were performed every 8 weeks accordingto RECIST 1.1.

Results

25 patients were enrolled (10 in EC 7 and 15 in EC 8): median age, 72.5y (range, 56-88y); median PS 1 (range, 0-1); 20 M:5F; median number ofprior systemic therapy regimens, 1 (range, 0-3). Median exposure toREGN2810 was 6 doses (range, 1- 22). The most common treatment-relatedadverse events of any grade were fatigue (16.7%), nausea, arthralgia,and rash (8.3% each). Each of the following Grade 3 related adverseevents (AEs) occurred once: AST elevation, ALT elevation, arthralgia,and rash.

Overall response rate (uPR+PR+CR) and disease control rate (ORR+SD) were48% (11/23; 3uPR, 5 PR, 2 CR, 1 uCR) and 70% (16/23, including 5 SD),respectively. Two patients are not yet evaluable. Median PFS and MedianOS are calculated, and only one patient has experienced PD duringREGN2810 treatment after initial response. Correlative science studiesare in process, including whole exome tumor DNA sequencing.

Conclusion

REGN2810 demonstrates robust antitumor activity in patients withadvanced CSCC.

Example 10 Clinical Trial of Anti-PD-1 Antibody Combined withHypofractionated Radiation Therapy Versus Standard Of Care In Patients65 Years Of Age With Newly Diagnosed Glioblastoma Introduction

Glioblastoma is a deadly disease with a median survival of approximately16 months in newly diagnosed patients (nGBM), and approximately 9 monthsin the recurrent setting (rGBM) (Friedman et al, 2009, J. Clin. Oncol.27: 4733-4740). The current standard of care for patients with newlydiagnosed glioblastoma is radiation (60 Gy over 6 weeks) with concurrenttemozolomide (TMZ) followed by adjuvant temozolomide (Stupp et al, 2005,N. Engl. J. Med. 352: 987-996), although subgroup analyses suggests thatthe addition of temozolomide may not improve efficacy in olderindividuals (Laperriere et al, 2013, Cancer Treat. Rev. 39: 350-357).

This Example describes a phase 3 study to evaluate efficacy of ananti-PD-1 antibody in combination with hypofractionated radiationtherapy (hfRT) versus standard of care (SoC) in terms of overallsurvival in patients 65 years old with nGBM.

The exemplary anti-PD-1 antibody used in this study is REGN2810 (alsoknown as H4H7798N as disclosed in US20150203579), a fully humanmonoclonal anti-PD-1 antibody comprising a heavy chain comprising theamino acid sequence of SEQ ID NO: 9 and a light chain comprising theamino acid sequence of SEQ ID NO: 10; an HCVR/LCVR amino acid sequencepair comprising SEQ ID NOs: 1 /2; and heavy and light chain CDRsequences comprising SEQ ID NOs: 3-8.

Study Objectives

The primary objective of the study is to evaluate efficacy in terms ofoverall survival (OS) of REGN2810 given in combination with hfRT versusstandard of care for patients 65 years old with nGBM.

The secondary objective of the study is to determine an improvement inprogression-free survival (PFS).

The other objectives of the study are: (i) improvement in Objectiveresponse rate (ORR), duration of response, and duration of diseasecontrol; (ii) clinical assessment using Neurologic Assessment inNeuro-Oncology (NANO) scale; (iii) safety; (iv) improvement in Qualityof life (QoL) and mental status; (v) changes in edema and steroid use;(vi) REGN2810 concentration in serum and anti-REGN2810 antibodies; and(vii) to explore potential pharmacodynamic, predictive or prognosticbiomarkers.

Study Design

This is a 2:1 randomized phase 3 study of REGN2810, a fully humanantibody to PD-1, combined with hypofractionated radiation therapyversus standard of care in patients 65 years of age with newly diagnosedglioblastoma. Patients are randomized to REGN2810 in combination withhypofractionated radiation therapy versus standard of care in a 2:1ratio with methylation status (methylated vs. unmethylated vs.undetermined) and extent of resection (partial vs. gross totalresection) as stratification factors. Efficacy is assessed by overallsurvival.

nGBM patients who are candidates for radiation therapy are randomized ina 2:1 ratio to receive one of the following treatments:

Investigational therapy: 3 mg/kg REGN2810 IV (every 2 weeks) plushypofractionated RT (6

Gy X 5, second week only). Radiation therapy is provided in Week 2 ofCycle 1, but not subsequent cycles. Comparator therapy: standard of careTMZ (oral, 75 mg/m², daily) in combination with standard RT (5 dailyradiation fractions/week of 2 Gy) for 6 weeks, followed by adjuvant TMZ(oral, 150 mg/m2 to 200 mg/m² 5 days/28 days) for 6 cycles. Radiationtherapy is provided in the first 6 week cycle only.

Study Duration

The study consists of a 28-day screening period, after which eligiblepatients may have up to twelve 56-day (8-week) treatment cycles for atotal of up to 96 weeks of treatment. During the screening period (day-28 to day -1), all eligible patients are required to have apre-treatment tumor resection available (partial or full resection) orbiospy for central pathology confirmation and MGMT methylationdetermination and confirmation.

After day 1/baseline, patients return to the clinic during cycle 1 ondays 8±3, 15±3, 29±3, 43±3, and 56±3. For each subsequent 8-week cycle(cycles 2-12), patients return to the clinic on days 1, 15±3, 29±3,43±3, and 56±3. Tumor assessments (brain MRI, iRANO and NANOassessments, MMSE, and EORTC QLQ-C30/BN20 questionnaires) are made atday 1/baseline and at the end of each treatment cycle. Extensive safetyevaluations occur on day 1 of each cycle; routine safety evaluationswill be conducted at each visit. Samples for assessment of biomarkers(cellular and molecular, described herein) related to REGN2810 treatmentexposure, clinical activity, or underlying disease are also collected.

During the 24-week follow-up period, patients return to the clinic 21 to42 days after the last study treatment for the first follow-up visit.Subsequent follow-up visits (follow-up visit 2 through follow-up visit7) occur every 28 days ±7 days. Tumor assessments (brain MRI, iRANO andNANO assessments, MMSE, and EORTC QLQ-C30/BN20 questionnaires) are madeat follow-up visit 3, follow-up visit 5, and follow-up visit 7.Extensive safety evaluations occur during the first follow-up visit;routine safety evaluations will be conducted at subsequent follow-upvisits. Samples for assessment of biomarkers (cellular and molecular,described herein) related to REGN2810 treatment exposure, clinicalactivity, or underlying disease are collected.

Target Population

The target population comprises patients 65 years old with nGBM.

Inclusion Criteria: A patient must meet the following criteria to beeligible for inclusion in the study: (1) newly diagnosed primaryglioblastoma with histological confirmation, cm in maximum diameter, whohas had partial or complete surgical resection; (2) Eastern CooperativeOncology Group (ECOG) performance status 0-2; (3) 65 years old; (4)Hepatic function: (a) Total bilirubin x upper limit of normal; (b) ALTand AST x ULN; (c) Alkaline phosphatase (ALP) x ULN; (5) Renal function:Serum creatinine x ULN; (6) Bone marrow function: Hemoglobin g/dL;Absolute neutrophil count (ANC) x 10⁹/L; Platelet count 75×10⁹/L; (7)Able to read, understand, and willing to sign the ICF; and (8) Abilityand willingness to comply with scheduled visits, treatment plans,laboratory tests, and other study-related procedures.

Exclusion Criteria: A patient who meets any of the following criteriawill be excluded from the study: (1) Any prior treatment for GBM (otherthan surgery); (2) Have known contraindication to Gd-MRI; (3) Ongoing orrecent (within 5 years) evidence of significant autoimmune disease thatrequired treatment with systemic immunosuppressive treatments, which maysuggest risk for immune-related adverse events (irAEs). The followingare not exclusionary: vitiligo, childhood asthma that has resolved,residual hypothyroidism that requires only hormone replacement, orpsoriasis that does not require systemic treatment.(4) Ongoing systemiccorticosteroid treatment, with the exception of corticosteroid use forother (non-tumor and non-immunosuppressive) indications up to a maximumof 10mg/day of prednisone or equivalent. (5) Primary tumors located inthe brainstem, spinal cord, or any secondary brain tumor activeinfection requiring therapy, including known infection with humanimmunodeficiency virus, or active infection with hepatitis B orhepatitis C virus. (6) History of pneumonitis within the last 5 years.(7) Any investigational or antitumor treatment within 30 days prior tothe initial administration of REGN2810. (8) History of documentedallergic reactions or acute hypersensitivity reaction attributed totreatment with antibody therapies in general, or to agents specificallyused in the study. (9) Inadequately controlled hypertension (defined assystolic blood pressure >150 mm Hg and/or diastolic blood pressure >100mm Hg) (10) Known allergy to doxycycline or tetracycline. (Precautiondue to presence of trace components in REGN2810.) (11) Prior history ofhypetensive crisis or hypertensive encephalophathy (12) History withinthe last 5 years of an invasive malignancy other than the one treated inthis study, with the exception of resected/ablated basal orsquamous-cell carcinoma of the skin or carcinoma in situ of the cervix,or other local tumors considered cured by local treatment. (13) Acute orchronic psychiatric problems that, under the evaluation of theinvestigator, make the patient ineligible for participation (14) Use ofNovocure Tumor Treating Fields (Optune NovoTTF-100A device) atscreening. Planned or anticipated use of Novocure Tumor Treating

Fields during study participation (15) Prior treatment with carmustinewafers (16) Continued sexual activity in men who are unwilling topractice adequate contraception during the study.

Study Treatments

Patients receive one of the following treatment regimens:

Investigational therapy: 3 mg/kg REGN2810 (administered IV infusion over30 minutes every 2 weeks for up to 96 weeks) plus hfRT in Week 2 ofCycle 1

Comparator: standard of care TMZ (oral, 75 mg/m2, daily) in combinationwith standard RT (5 daily radiation fractions/week of 2 Gy) for 6 weeks,followed by adjuvant TMZ (oral, 150 mg/m2 to 200 mg/m² 5 days/28 days)for 6 cycles. Radiation therapy is provided in the first cycle only.

REGN2810 is supplied as a liquid in sterile, single-use vials. Each vialcontains a volume sufficient to withdraw 10 mL of REGN2810 at aconcentration of 25 mg/mL. REGN2810 is administered as a 30 minute IVinfusion. Each patient's dose will depend on individual body weight. Thedose of REGN2810 must be adjusted each cycle for changes in body weightof 10c)/0.

Radiation Therapy: Patients in the control arm receive standardradiotherapy (60 Gy over 6 weeks). Patients in the experimentaltreatment group receive hfRT (6 Gy X 5 daily fractions) administered 1week after the first dose of REGN2810.

REGN2810 plus Radiation (Investigational Treatment): REGN2810 isadministered by IV infusion over 30 minutes every 14 days for 96 weeksin combination with hfRT from day 8 to day 12.

Planned combination REGN2810 and hfRT regimen: 3 mg/kg REGN2810 infusionover 30 minutes every 14 days for 96 weeks plus radiation therapy (hfRTat 6 Gy x 5 daily fractions; given 1 week after the first dose ofREGN2810, preferably on consecutive days).

Specifications for Radiation Therapy: Patients receive 30 Gy given as 5fractions of 6 Gy administered daily starting 1 week after the firstdose of REGN2810.

Comparator Arm: Standard of Care: TMZ (oral, 75 mg/m², daily) incombination with standard RT (5 daily radiation fractions/week of 2 Gy)for 6 weeks, followed by adjuvant oral TMZ. The dose of TMZ is 150 mg/m²for the first 5 days of the first adjuvant cycle, and is increased 200mg/m² for 5 days/28 days starting with the second cycle if there is nounacceptable hematologic toxicities with the first adjuvant cycle.

If, during the first adjuvant cycle, all non-hematologic toxicitiesobserved are grade 2 (except alopecia, nausea and vomiting) andplatelets are 100×109/L and ANC >=1.5×109/L, then the TMZ dose should beescalated to dose level 1 (200 mg/m²) and this dose should be used asthe starting dose for subsequent cycles. If after cycle 1 TMZ has to bedelayed because of ongoing non-hematologic toxicities of grade 2, thenno escalation is possible. If the dose was not escalated at the secondcycle, then the dose should not be escalated in subsequent cycles.

Treatments for CNS Edema: Any patient who develops symptomaticintracranial edema during the study has REGN2810 dosing and radiationtherapy held until the edema subsides.

For patients who develop intracranial edema, bevacizumab is administeredIV, as needed (PRN), at a reduced dose from the standard (suggested doseof 5 mg/kg Q2W for up to 3 doses, not more than 10 mg/kg Q2W per dose),unless contraindicated (e.g., unless the patient had surgery within thepast 28 days).

If bevacizumab does not resolve the intracranial edema, systemiccorticosteroids, in addition to or as replacement for bevacizumab, atthe lowest dose deeded to be appropriate for symptom management may beadministered. For patients who are bevacizumab intolerantcorticosteroids are used at a dose deeded to be appropriate for symptommanagement.

Study Variables

The primary efficacy endpoint is overall survival (OS), which is definedas the time interval from the date of randomization to the date of deathdue to any cause.

The key secondary endpoint is progression free survival (PFS), which isdefined as the time interval from the date of randomization to the dateof first observation of disease progression or the date of death (due toany cause). Disease progression is determined by iRANO criteria.

The other secondary efficacy endpoints are:

Objective response rate (ORR): defined as the proportion of patientswith confirmed complete response (CR) or confirmed partial response(PR), defined by Immunotherapy Response Assessment in Neuro-Oncology(iRANO) criteria relative to the total number of patients in theanalysis population.

Duration of response: determined for patients with best overall responseof CR or PR. Duration of response is measured from the time measurementcriteria are first met for CR/PR (whichever is first recorded) until thefirst date of recurrent or progressive disease (radiographic), or deathdue to any cause.

Duration of disease control: determined for patients with best overallresponse of SD, CR, or PR. Duration of disease control is measured fromthe start of treatment until the first date of recurrent or progressivedisease (radiographic), or death due to any cause.

Quality of Life and Symptom Control Variables: The quality of life andsymptom control variables are:

Five functional scales, three symptom scales, one global measure ofhealth status and six single-item scales assessing symptoms using theEORTC QLQ-C30 questionnaires during the study

Four scales and seven single items using the EORTC QLQ-BN20questionnaires during the study

Clinical assessment using NANO;

The total score of the MMSE during the study

Use of corticosteroid at baseline, cumulative corticosteroid use duringthe study, and the duration of steroid-free or low dose steroid useduring the progression-free period of study

Use of bevacizumab PRN at baseline, cumulative bevacizumab PRN duringthe study, and the duration of bevacizumab-free during theprogression-free period of study

Exploratory Biomarker Variables: Other endpoint includespharmacodynamic, prognostic, and predictive biomarkers related toclinical response, mechanism of action, and possible AEs associated withREGN2810 after treatment. The biomarker variables include:

Expression levels of immune checkpoint receptors PD-L1, GITR, and LAG3,as well as other potential biomarkers (e.g., EGFRvIll, Ki67, etc) intumor samples;

Number and distribution of TI Ls in tumor samples;

IDH1 mutational status, microsatelite instabilty (MSI), and mutationalburden in tumor samples;

Circulating biomarkers including cytokines and angiogenic factors;

Cell subsets and expression levels of biomarkers of interest in PBMCs;

MGMT promoter methylation status (also used for stratification)

Other variables include REGN2810 concentration in serum (pharmacokineticvariables) and development of anti-REGN2810 antibodies.

Procedures and Assessments

After a screening period of up to 28 days, patients receive up to twelve56-day treatment cycles for a total of up to 96 weeks of treatment,followed by a 24 week follow-up period. Efficacy, safety, PK, ADA, andexploratory biomarker analysis are performed.

Efficacy Procedures

MRI: An MRI for tumor assessment is performed 72 hrs post-surgery, atthe screening visit (within 28 days prior to infusion), on day 56±3 ofevery cycle (approximately every 8 weeks), and when PD is suspected.Patients for whom disease has not progressed have additional tumorassessments performed at follow-up visits 3, 5, and 7. Note: if PD hasbeen confirmed, additional scans will not be required during follow-upvisits. If pre and post- surgery MRIs were performed prior to enrollmentonto the study, those scans must also be submitted to the study to aidin determination of tumor volume and tumor progression.

Tumor response evaluation is performed according to iRANO; and clinicalneurologic assessment will be performed by NANO. Assessments accordingto RANO are also performed as a supportive exploration; however, theprimary determination of disease progression for an individual patientis made according to iRANO.

The European Organization for Research and Treatment of Cancer Qualityof Life Questionnaire (EORTC QLQ-C30) and the EORTC Brain Cancer Module(EORTC QLQ-BN20) Questionnaire: The EORTC QLQ-C30 is a 30-itemquestionnaire that assesses health-related quality of life (HRQoL) incancer patients with 15 scales (single- or multi-item), each withpossible scores ranging from 0 to 100. Of the 30 items, 24 aggregateinto 9 multi-item scales representing various HRQoL dimensions: 5functioning scales (physical, role, emotional, cognitive, and social), 3symptom scales (fatigue, pain, and nausea), and 1 global measure ofhealth status. The remaining 6 single-item scales assess symptoms:dyspnea, appetite loss, sleep disturbance, constipation and diarrhea,and the perceived financial impact of the disease treatment. High scoresindicate better HRQoL for the global measure of health status andfunctioning scales, and worse HRQoL for the symptom scales.

The EORTC QLQ-BN20 is a 20-item QoL assessment specific to brainneoplasms and is intended to supplement the EORTC QLQ-C30 when assessinghealth-related quality of life. The EORTC QLQ-BN20 questionnaireassesses disease symptoms, side-effects of treatment, and some specificpsychosocial issues of importance to patients with brain cancer using 4scales (assessing future uncertainty, visual disorder, motordysfunction, and communication deficit) and 7 single items (assessingother disease symtpoms [eg, headaches and seizures] and treatment toxiceffects [e.g., hair loss]). The possible scores range from 0 to 100;high scores indicate worse HRQoL.

Mini-Mental Status Assessment: The Mini-Mental State Examination (MMSE©)is a brief, quantitative measure of cognitive status in adults. It canbe used to screen for cognitive impairment, to estimate the severity ofcognitive impairment at a given point in time, and to follow the courseof cognitive changes in an individual over time. In this study, the MMSEscore is part of the neurological examination performed in the contextof the disease assessments.

MMSE is performed at day 1/baseline, at the end of every treatmentcycle, and every 8 weeks during the follow-up period. The MMSEassessments coincide with the schedule of disease assessments, but theymust be completed prior to announcing the radiological assessment resultto the patient. The MMSE may be completed at the beginning of the nextscheduled treatment administration. During survival follow-up period,the MMSE should continue to be completed at every second survival visit(every 8 weeks) if the patient has not yet progressed.

The total score of the MMSE has a possible range from 0 (worst) to 30(best).

Safety Procedures

At cycle 1 day 1 and on all subsequent treatment days, vital signs,including temperature, resting blood pressure, pulse, and respiration,along with weight will be collected prior to infusion, and approximately15 minutes after the completion of the infusion. A complete physicalexamination and a 12-lead ECG is carried out at the beginning of everycycle.

Exploratory Tumor Biomarker Procedures

The biomarkers of interest that are analyzed by immunohistochemistry(IHC) include but are not limited to EGFRvIll and biomarkers of cellproliferation (for example, Ki67). Expression levels (mRNA and/orprotein) of PD-L1, GITR, and LAG-3, as well as lineage markers of tumorinfiltrating lymphocytes (CD4, CD8, CD25, FoxP3) are analyzed in tumorbiopsy samples to explore potential effect of REGN2810.

Tumor tissue samples may be used for extraction of tumor DNA and RNA andsubsequent analyses of putative genetic biomarkers relevant to studytreatment and glioblastoma. A blood sample is collected for isolation ofgerm-line DNA on day 1/baseline (predose), or at any study visit, ifcollection at day 1/baseline is not possible. Analyses of the tumor DNAinclude (but are not limited to) methylation status of MGMT promoter,IDH1 mutational status, microsatelite instabilty (MSI), and tumormutation burden (which both may be predictive of response to REGN2810and other immunotherapeutic agents). Analysis of genetic variants intumor (somatic) DNA and germ-line DNA that may affect diseaseprogression, drug response and possible toxicities are performed.Germ-line DNA is also used for comparison to tumor DNA to explorepotential novel genetic variants underlying malignant processes.

Results

REGN2810 in combination with hfRT is safe and well-tolerated by patientswith nGBM. Administration of REGN2810 in combination with hfRT inhibitstumor growth and/or promotes tumor regression in patients with nGBM ascompared to standard of care therapy. Patients with nGBM treated withREGN2810 and hfRT show a longer OS as compared to standard of caretherapy.

Example 11 Clinical Trial of REGN2810 in Patients with AdvancedCutaneous Squamous Cell Carcinoma

This Example describes a phase 2 trial that was conducted to confirm thepositive results seen in patients with advanced CSCC in a phase 1 trial(see Examples 7, 8 and 9 herein)

Study Objectives

The primary objective of this study is to estimate the clinical benefitof REGN2810 monotherapy for patients with metastatic (nodal or distant)cutaneous squamous cell carcinoma (CSCC) (Group 1) or with unresectablelocally advanced CSCC (Group 2), as measured by overall response rate(ORR).

The secondary objectives of the study are: (i) to estimate ORR; (ii) toestimate the duration of response, progression-free survival (PFS), andoverall survival (OS); (iii) to estimate the complete response (CR)rate; (iv) to assess the safety and tolerability of REGN2810; (v) toassess the pharmacokinetics (PK) of REGN2810; (vi) to assess theimmunogenicity of REGN2810; and (vii) to assess the impact of REGN2810on quality of life using EORTC QLQ-030.

Study Design

This is a phase 2, non-randomized, 2-group, multicenter study ofREGN2810 at a dose of 3 mg/kg administered intravenously (IV) every 2weeks for patients with advanced CSCC. The study has 2 groups. Group 1is for patients with metastatic CSCC. Group 2 is for patients withunresectable locally advanced CSCC. All patients undergo screeningprocedures to determine eligibility within 28 days prior to the initialadministration of REGN2810.

After a screening period of up to 28 days, patients receive up to twelve56-day (8-week) treatment cycles for up to 96 weeks of treatment. Eachpatient receives 3 mg/kg REGN2810 IV on days 1, 15±3, 29±3, and 43±3during each treatment cycle. Tumor assessments are made at the end ofeach treatment cycle. Extensive safety evaluations occur on day 1 ofeach cycle, with routine safety evaluations to be conducted at eachREGN2810 dosing visit.

A patient receives treatment until the 96-week treatment period iscomplete, or until disease progression, unacceptable toxicity,withdrawal of consent, or confirmed CR. Patients with confirmed CR aftera minimum of 48 weeks of treatment may elect to discontinue treatmentand continue with all relevant study assessments (e.g., efficacyassessments).

Study Duration

Screening (up to 4 weeks), up to 96 weeks of treatment, and up to 6months of follow-up. Study Population

Patients with metastatic CSCC or with unresectable locally advanced CSCCStudy Treatment

REGN2810 3 mg/kg administered IV over 30 minutes every 14 days for 96weeks Study Variables

The primary efficacy endpoint for this study is ORR during the 12treatment cycles. Overall response rate is assessed separately forpatients with metastatic CSCC or unresectable locally advanced CSCC: Forpatients in Group 1, Response Evaluation Criteria in Solid Tumors(RECIST) version 1.1 is used to determine ORR. For patients in Group 2,composite response criteria are used to determine ORR. In patientsachieving a CR, tumor biopsies are used in the final determination ofcomplete versus partial response (PR).

The secondary efficacy outcome measures are: duration of response;duration of disease control; PFS; OS; CR rate; change in scores ofpatient-reported outcomes on EORTC QLQ-C30; adverse events (AEs);concentrations of REGN2810 in serum; and anti-REGN2810 antibodies.

Procedures and Assessments

Tumor imaging (computed tomography [CT] or magnetic resonance imaging[MRI]) and digital medical photography (for externally visible lesions)is performed to measure tumor burden and to characterize the efficacyprofile of study treatments using response criteria.

Physical examination, laboratory tests, vital signs, electrocardiogram(ECG), pregnancy test for women of childbearing potential, and recordingof AEs and concomitant medications are performed to ensure patientsafety and to characterize the safety profiles of study treatments.

Other assessments include: Peripheral blood samples for PK; Peripheralblood samples to assess anti-REGN2810 antibodies; Tumor biopsies; andQuality of life assessments.

Results

The trial is fully enrolled and results to-date are in line with phase Iresults (described herein in Examples 7, 8 and 9) with patients showinginhibition of tumor growth upon administration of REGN2810. Patientswith metastatic LSCC who have been treated with prior therapies and arenot amenable to surgery show complete response, partial response orstable disease on treatment with anti-PD-1 antibody REGN2810.

Example 12 Clinical Trial of REGN2810 in Patients with Advanced BasalCell Carcinoma

A phase 2 trial was conducted to confirm the positive results seen inpatients with advanced BCC in a phase 1 trial (see Examples 7 and 8herein).

Study Objectives

The primary objective of the study is to estimate the overall responserate (ORR) for metastatic basal cell carcinoma (BCC) (Group I) orunresectable locally advanced BCC (Group II), when treated with REGN2810monotherapy in patients who have progressed on Hedgehog pathwayinhibitor (HHI) or were intolerant of prior HHI therapy.

The secondary objectives for both Group I and Group II are to: (i)estimate ORR according to investigator review; (ii) estimate theduration of response, progression-free survival (PFS) and overallsurvival (OS); (iii) estimate the complete response (CR) rate; (iv)assess the safety and tolerability of REGN2810; (v) assess thepharmacokinetics (PK) of REGN2810; (vi) assess the immunogenicity ofREGN2810; and (vii) assess the impact of REGN2810 on quality of lifeusing

European Organisation for Research and Treatment of Cancer Quality ofLife Questionnaire Core 30 (EORTC QLQ-C30) and Skindex-16.

Study Design

This is a phase 2, non-randomized, 2-group, multi-center study ofREGN2810 at a 350 mg dose administered intravenously (IV) every 3 weeks(Q3VV) in patients with advanced BCC who experienced progression ofdisease on HHI therapy, or were intolerant of prior HHI therapy. Thestudy has 2 groups. Group 1 is for patients with metastatic BCC. Group 2is for patients with unresectable locally advanced BCC. All patientsundergo screening procedures to determine eligibility within 28 daysprior to the initial administration of REGN2810. There is norandomization or placebo control.

After a screening period of up to 28 days, patients receive up to 93weeks of treatment. Each patient receives a 350 mg Q3W dose of REGN2810IV. The infusion time for REGN2810 is approximately 30 minutes (±10minutes). Tumor assessments are made at the end of each treatment cycle,5 treatment cycles of 9 weeks followed by 4 treatment cycles of 12weeks). Extensive safety evaluations occur on day 1 of each cycle, withroutine safety evaluations to be conducted at each REGN2810 dosingvisit.

A patient receives treatment until the 93-week treatment period iscomplete, or until disease progression (PD), unacceptable toxicity,withdrawal of consent, or confirmed CR. Patients with confirmed CR aftera minimum of 48 weeks of treatment may elect to discontinue treatmentand continue with all relevant study assessments (e.g., efficacyassessments). Patients who discontinue study treatment due to PD returnto the clinic 30 days (range: 28 days to 42 days) after the last studytreatment to complete the end-of-study (EOS) assessments. After the EOSvisit, patients are followed for survival status until death, loss tofollow up, or study termination.

Study Duration

After a screening period of up to 28 days, patients receive up to 93weeks of treatment. After the end of study visit, there is a follow-upperiod consisting of periods of 28 days. Patients are followed forsurvival status until death, loss to follow up, or study termination.

Study Population

Patients with metastatic (Group 1) or unresectable locally advanced(Group 2) BCC who experienced progression of disease on HHI therapy, orwere intolerant of prior HHI therapy.

Study Treatment

Study treatment comprised 350 mg REGN2810 administered IV over 30minutes (±10 minutes) once every 3 weeks (q3w) for up to 93 weeks.

Endpoints

The primary efficacy endpoint for this study is the ORR. The ORR isassessed separately for patients with metastatic BCC (Group 1) orunresectable locally advanced BCC (Group 2):

For patients in Group 1 (metastatic BCC), Response Evaluation Criteriain Solid Tumors

(RECIST) version 1.1 is used to determine ORR. Clinical responsecriteria may be used for patients with externally visible targetlesions, if all metastatic lesions are not measurable by RECIST (as mayoccur in patients with bone-only metastases).

For patients in Group 2 (unresectable locally advanced BCC), clinicalcriteria are used to determine ORR. Composite response criteria are usedfor patients with lesions that are measureable by both clinical responsecriteria and RECIST 1.1.

The secondary endpoints are: (i) Duration of response; (ii) PFS; (iii)OS; (iv) CR rate; (v) Change in scores of patient-reported outcomes inthe EORTC QLQ-C30 and the Skindex-16; (vi) Adverse events (AEs); (vii)Concentrations of REGN2810 in serum; and (viii) Anti-REGN2810antibodies.

Procedures and Assessments

Tumor imaging (computed tomography [CT] or magnetic resonance imaging[MRI]) and digital medical photography (for externally visible lesions)are performed to measure tumor burden and to characterize the efficacyprofile of study treatments using response criteria. Physicalexamination, laboratory tests, vital signs, electrocardiogram (ECG),pregnancy test for women of childbearing potential, and recording of AEsand concomitant medications are performed to ensure patient safety andto characterize the safety profiles of study treatments. Otherassessments include blood samples for PK, blood samples to assessanti-REGN2810 antibodies, tumor biopsies, biomarkers, and quality oflife assessments. Results

It is expected that consistent with phase 1 results (see Examples 7 and8 herein) administration of REGN2810 will lead to tumor regression inpatients with advanced basal cell carcinoma who showed progression ofdisease upon treatment with a Hedgehog pathway inhibitor (HHI) or wereintolerant of prior HHI therapy. Patients show complete response,partial response or stable disease upon treatment with REGN2810.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

SEQUENCE LISTING

The sequence listing of the present application is submittedelectronically as a ST.26 formatted sequence listing with a file name“00530_Seq-Listing.xml,” a creation date of Sep. 17, 2022, and a size of12.7 KB. The sequence listing submitted is part of the specification andis hereby incorporated by reference in its entirety. Sequences disclosedherein and having a length that is below the minimum length permittedunder ST.26 format are provided in the table below:

SEQ ID NO. Sequence 7 Ala Ala Ser

1. A method of treating or inhibiting the growth of a tumor comprising:(a) selecting a patient with basal cell carcinoma (BCC); and (b)administering to the patient a therapeutically effective amount of anantibody or antigen-binding fragment thereof that specifically bindsPD-1; wherein the antibody or antigen-binding fragment thereof comprisesthe three heavy chain complementarity determining regions (HCDR1, HCDR2and HCDR3) of a heavy chain variable region (HCVR) comprising the aminoacid sequence of SEQ ID NO: 1 and three light chain complementaritydetermining regions (LCDR1, LCDR2 and LCDR3) of a light chain variableregion (LCVR) comprising the amino acid sequence of SEQ ID NO:
 2. 2-30.(canceled)
 31. The method of claim 1, wherein the BCC is metastatic,unresectable and/or locally advanced.
 32. The method of claim 1, whereinthe patient was previously treated with at least one anti-cancertherapy.
 33. The method of claim 32, wherein the anti-cancer therapy isselected from surgery, radiation, chemotherapy, a hedgehog pathwayinhibitor, and another anti-PD-1 antibody.
 34. The method of claim 1,wherein the BCC has progressed after prior treatment with, or thepatient is intolerant to, a hedgehog pathway inhibitor.
 35. The methodof claim 34, wherein the hedgehog pathway inhibitor is selected fromvismodegib and sonedegib.
 36. The method of claim 1, wherein the BCC isinoperable or the patient is not amenable to curative surgery,radiation, or treatment with a hedgehog pathway inhibitor.
 37. Themethod of claim 1, wherein the antibody or antigen-binding fragmentthereof is administered in one or more doses, wherein each dose isadministered 0.5 to 4 weeks after the immediately preceding dose. 38.The method of claim 37, wherein each dose is administered 2 weeks afterthe immediately preceding dose.
 39. The method of claim 37, wherein eachdose is administered 3 weeks after the immediately preceding dose. 40.The method of any one of claims 37, wherein each dose comprises 1, 3, or10 mg/kg of patient's body weight.
 41. The method of claim 40, whereineach dose comprises 3 mg/kg of the patient's body weight.
 42. The methodof claim 37, wherein each dose comprises 50-600 mg of the antibody orantigen-binding fragment thereof.
 43. The method of claim 42, whereineach dose comprises 200, 250, or 350 mg of the antibody orantigen-binding fragment thereof
 44. The method of claim 1, wherein thepatient is resistant or inadequately responsive to, or relapsed afterprior therapy.
 45. The method of claim 1, wherein the antibody orantigen-binding fragment thereof is administered as a monotherapy. 46.The method of claim 1, wherein the administration leads to at least oneeffect selected from inhibition of tumor growth, tumor regression,reduction in the size of a tumor, reduction in tumor cell number, delayin tumor growth, abscopal effect, inhibition of tumor metastasis,reduction in metastatic lesions over time, reduced use ofchemotherapeutic or cytotoxic agents, reduction in tumor burden,increase in progression-free survival, increase in overall survival,complete response, partial response, and stable disease.
 47. The methodof claim 1, further comprising administering to the patient anadditional therapeutic agent or therapy selected from a hedgehog pathwayinhibitor, surgery, radiation, a chemotherapeutic agent, a cancervaccine, a programmed death ligand 1 (PD-L1) inhibitor, a lymphocyteactivation gene 3 (LAG3) inhibitor, a cytotoxic T-lymphocyte-associatedprotein 4 (CTLA-4) inhibitor, an anti-glucocorticoid-induced tumornecrosis factor receptor (GITR) antibody, a T-cell immunoglobulin andmucin-domain containing-3 (TIM3) inhibitor, a B- and T-lymphocyteattenuator (BTLA) inhibitor, a T cell immunoreceptor with Ig and ITIMdomains (TIGIT) inhibitor, a CD47 inhibitor, anindoleamine-2,3-dioxygenase (IDO) inhibitor, a bispecificanti-CD³/_(a)nti-CD20 antibody, a vascular endothelial growth factor(VEGF) antagonist, an angiopoietin-2 (Ang2) inhibitor, a transforminggrowth factor beta (TGF(3) inhibitor, a CD38 inhibitor, an epidermalgrowth factor receptor (EGFR) inhibitor, granulocyte-macrophagecolony-stimulating factor (GM-CSF), cyclophosphamide, an antibody to atumor-specific antigen, Bacillus Calmette-Guerin vaccine, a cytotoxin,an interleukin 6 receptor (IL-6R) inhibitor, an interleukin 4 receptor(IL-4R) inhibitor, an IL-10 inhibitor, IL-2, IL-7, IL-21, IL-15, anantibody-drug conjugate, an anti-inflammatory drug, and a dietarysupplement.
 48. The method of claim 1, wherein the antibody orantigen-binding fragment thereof is administered intravenously,subcutaneously, or intraperitoneally.
 49. The method of claim 1, whereinHCDR1 comprises the amino acid sequence of SEQ ID NO: 3; HCDR2 comprisesthe amino acid sequence of SEQ ID NO: 4; HCDR3 comprises the amino acidsequence of SEQ ID NO: 5; LCDR1 comprises the amino acid sequence of SEQID NO: 6; LCDR2 comprises the amino acid sequence of SEQ ID NO: 7; andLCDR3 comprises the amino acid sequence of SEQ ID NO:
 8. 50. The methodof claim 1, wherein the antibody or antigen-binding fragment thereofcomprises a HCVR comprising the amino acid sequence of SEQ ID NO: 1 andthe LCVR comprising the amino acid sequence of SEQ ID NO:
 2. 51. Themethod of claim 1, wherein the antibody or antigen-binding fragmentthereof comprises a HCVR with 90% sequence identity to SEQ ID NO:
 1. 52.The method of claim 1, wherein the antibody or antigen-binding fragmentthereof comprises a LCVR with 90% sequence identity to SEQ ID NO:
 2. 53.The method of claim 1, wherein the antibody or antigen-binding fragmentthereof comprises a HCVR with 90% sequence identity to SEQ ID NO: 1 anda LCVR with 90% sequence identity to SEQ ID NO:
 2. 54. The method ofclaim 1, wherein the antibody comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 9 and a light chain comprising theamino acid sequence of SEQ ID NO:
 10. 55. A method of treating orinhibiting the growth of a tumor comprising: (a) selecting a patientwith locally advanced basal cell carcinoma (BCC) or metastatic BCC,wherein the patient was previously treated with a hedgehog pathwayinhibitor or is not amenable to treatment with a hedgehog pathwayinhibitor; and (b) administering to the patient a dose of 350 mg of anantibody that specifically binds PD-1, wherein the dose is administeredintravenously every three weeks; wherein the antibody comprises threeheavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3)of a heavy chain variable region (HCVR) comprising the amino acidsequence of SEQ ID NO: 1, and three light chain complementaritydetermining regions (LCDR1, LCDR2 and LCDR3) comprising the amino acidsequence of SEQ ID NO:
 2. 56. The method of claim 55, wherein HCDR1comprises the amino acid sequence of SEQ ID NO: 3; HCDR2 comprises theamino acid sequence of SEQ ID NO: 4; HCDR3 comprises the amino acidsequence of SEQ ID NO: 5; LCDR1 comprises the amino acid sequence of SEQID NO: 6; LCDR2 comprises the amino acid sequence of SEQ ID NO: 7; andLCDR3 comprises the amino acid sequence of SEQ ID NO:
 8. 57. The methodof claim 55, wherein the antibody comprises a HCVR comprising the aminoacid sequence of SEQ ID NO: 1 and a LCVR comprising the amino acidsequence of SEQ ID NO:
 2. 58. The method of claim 55, wherein theantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO: 9 and a light chain comprising the amino acid sequence of SEQID NO: 10.